Alpha-amylase variants and polynucleotides encoding same

ABSTRACT

The present invention relates to alpha-amylase variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/346,107 filed on Mar. 20, 2014, now abandoned, which is a 35 U.S.C.371 national application of PCT/EP2012/070578 filed Oct. 17, 2012, whichclaims priority or the benefit under 35 U.S.C. 119 of Europeanapplication no. 11185479.0 filed Oct. 17, 2011 and U.S. provisionalapplication No. 61/548,416 filed Oct. 18, 2011, the contents of whichare fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference. The name of the filecontaining the Sequence Listing is SQ.txt, which was created on Sep. 25,2018 and has 61.7 kB. The file corresponds to the Sequence Listingoriginally filed on Mar. 20, 2014 in parent application Ser. No.14/346,107.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to alpha-amylase variants, polynucleotidesencoding the variants, methods of producing the variants, and methods ofusing the variants.

Description of the Related Art

Alpha-amylases (E.C. 3.2.1.1) constitute a group of enzymes whichcatalyze hydrolysis of starch, glycogen and related polysaccharides andoligosaccharides.

Alpha-amylases are used commercially for a variety of purposes such asin the initial stages of starch processing (e.g., liquefaction); in wetmilling processes; and in alcohol production from carbohydrate sources.They are also used as cleaning agents or adjuncts in detergent matrices;in the textile industry for starch desizing; in baking applications; inthe beverage industry; in oil fields in drilling processes; in recyclingprocesses, e.g., for de-inking paper; and in animal feed.

Some commercial alpha-amylases for, e.g., starch liquefaction originatefrom Bacillus licheniformis or Bacillus stearothermophilus. Proteinengineered variants of wild type enzymes have been developed to overcomeprocess issues. There is still a need, though, for novel alpha-amylaseswith improved properties, such as higher stability at low pH, lowcalcium and high temperature. Such enzymes will allow the starchliquefaction process to be run at reduced pH which has a positiveinfluence on chemical savings.

It is an object of the present invention to provide novel alpha-amylasevariants having an increased stability at low pH and/or at hightemperature, in particular at low calcium concentrations.

SUMMARY OF THE INVENTION

The present inventors have found that alpha-amylase variants comprisingan alteration at one or more positions corresponding to any of positions1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213,239, 279, 316, 318, 360, 416, 437 and 450 of SEQ ID NO: 1 have anincreased stability when incubated at low pH and/or at high temperature,in particular at low calcium concentrations, and in particular in thepresence of at least 0.1% starch, e.g., in the presence of 0.9% or 1%starch.

The present invention therefore relates to alpha-amylase variantscomprising an alteration at one or more positions corresponding to anyof positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176, 185, 201,205, 213, 239, 279, 316, 318, 360, 416, 437 and 450 of SEQ ID NO: 1,wherein the variant has at least 60% and less than 100% sequenceidentity to (i) the mature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQID NO: 1, amino acids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485 ofSEQ ID NO: 3, amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484of SEQ ID NO: 5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to485 of SEQ ID NO: 7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1to 485 of SEQ ID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10, aminoacids 1 to 485 of SEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12,amino acids 1 to 483 of SEQ ID NO: 13 or amino acids 1 to 481 of SEQ IDNO: 14, and wherein the variant has alpha-amylase activity.

The present invention also relates to isolated polynucleotides encodingthe variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the variants.

The present invention also relates to uses of the variants of theinvention and to a method of producing liquefied starch and a method ofproducing a fermentation product.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of alpha-amylases with the amino acidsequences of:

SEQ ID NO: 1 is a Bacillus licheniformis alpha-amylase.

SEQ ID NO: 2 is a Bacillus stearothermophilus alpha-amylase.

SEQ ID NO: 3 is the Bacillus alpha-amylase TS-23 described in J. Appl.Microbiology, 1997, 82: 325-334 (SWALL:q59222).

SEQ ID NO: 4 is Bacillus flavothermus alpha-amylase AMY1048 described inWO 2005/001064.

SEQ ID NO: 5 is Bacillus alpha-amylase TS-22 described as SEQ ID NO: 21in WO 04/113511.

SEQ ID NO: 6 is a Bacillus amyloliquefaciens alpha-amylase.

SEQ ID NO: 7 is Bacillus alkaline sp. SP690 amylase described as SEQ IDNO 1 in WO 95/26397.

SEQ ID NO: 8 is Bacillus halmapalus alpha-amylase described as SEQ ID NO2 in WO 95/26397.

SEQ ID NO: 9 is Bacillus alkaline sp. AA560 amylase described as SEQ IDNO 4 in WO 00/60060.

SEQ ID NO: 10 is Bacillus alkaline sp. A 7-7 amylase described as SEQ IDNO 2 in WO200210356.

SEQ ID NO: 11 is Bacillus alkaline sp. SP707 amylase described inTsukamoto et al., 1988, Biochem. Biophys. Res. Commun. 151: 25-33).

SEQ ID NO: 12 is Bacillus alkaline sp. K-38 amylase described as SEQ IDNO 2 in EP 1022334.

SEQ ID NO: 13 is a Bacillus licheniformis alpha-amylase described in Leeet al, 2006, J. Biochem, 139: 997-1005.

SEQ ID NO: 14 is a variant alpha-amylase LE399 previously disclosed in,e.g., WO 2002/010355.

Definitions

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-amylase: Alpha-amylases (E.C. 3.2.1.1) are a group of enzymeswhich catalyze the hydrolysis of starch and other linear and branched1,4 glucosidic oligo- and polysaccharides. The skilled person will knowhow to determine alpha-amylase activity. It may be determined accordingto the procedure described in the Examples, e.g., by the PNP-G7 assay.In one aspect, the variants of the present invention have at least 20%,e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 100% of the alpha-amylaseactivity of the mature polypeptide of SEQ ID NO: 1. In another aspect, avariant of the present application has at least 20%, e.g., at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 100% of the alpha-amylase activity of its parent.

-   -   cDNA: The term “cDNA” means a DNA molecule that can be prepared        by reverse transcription from a mature, spliced, mRNA molecule        obtained from a eukaryotic or prokaryotic cell. cDNA lacks        intron sequences that may be present in the corresponding        genomic DNA. The initial, primary RNA transcript is a precursor        to mRNA that is processed through a series of steps, including        splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has alpha-amylaseactivity. In one aspect, a fragment contains at least 300 amino acidresidues, at least 350 amino acid residues, at least 400 amino acidresidues, at least 450 amino acid residues, at least 470 amino acidresidues, or at least 480 amino acid residues.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. It is known in the art that a hostcell may produce a mixture of two or more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide. The mature form of somealpha-amylases, e.g., some bacterial alpha-amylases, comprises acatalytic domain containing the active site for substrate hydrolysis andone or more carbohydrate-binding modules (CBM) for binding to thecarbohydrate substrate (starch) and optionally a polypeptide linking theCBM(s) with the catalytic domain, a region of the latter type usuallybeing denoted a “linker”.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent alpha-amylase: The term “parent” or “parentalpha-amylase” means an alpha-amylase to which an alteration is made toproduce the enzyme variants of the present invention. The parent may bea naturally occurring (wild-type) polypeptide or a variant or fragmentthereof.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

Variant: The term “variant” means a polypeptide having alpha-amylaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding one or more (e.g.,several) amino acids, e.g., 1-5 amino acids, adjacent to the amino acidoccupying a position. In one aspect, the variants of the presentinvention have at least 20%, e.g., at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least100% of the alpha-amylase activity of the mature polypeptide of SEQ IDNO: 1. In another aspect, a variant of the present application has atleast 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, or at least 100% of thealpha-amylase activity of its parent. The alpha-amylase activity may bedetermined by the PNP-G7 assay described in the Examples.

Wild-type alpha-amylase: The term “wild-type” alpha-amylase means analpha-amylase expressed by a naturally occurring microorganism, such asa bacterium, yeast, or filamentous fungus found in nature.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 1 is used to determine the corresponding amino acidresidue in another alpha-amylase. The amino acid sequence of anotheralpha-amylase is aligned with the mature polypeptide disclosed in SEQ IDNO: 1, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the mature polypeptidedisclosed in SEQ ID NO: 1 is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277), preferably version 5.0.0 or later. Theparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anotheralpha-amylase can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010,Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 1 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions.

For an amino acid substitution, the following nomenclature is used:Original amino acid, position, substituted amino acid. Accordingly, thesubstitution of threonine at position 226 with alanine is designated as“Thr226Ala” or “T226A”. Multiple mutations are separated by additionmarks (“+”), e.g., “Gly205Arg+Ser411Phe” or “G205R+S411F”, representingsubstitutions at positions 205 and 411 of glycine (G) with arginine (R)and serine (S) with phenylalanine (F), respectively. In the Examples ofthe present application, multiple mutations are separated by a space,e.g., G205R S411F representing G205R+S411F.

Deletions.

For an amino acid deletion, the following nomenclature is used: Originalamino acid, position, *. Accordingly, the deletion of glycine atposition 195 is designated as “Gly195*” or “G195*”. Multiple deletionsare separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or“G195*+S411*”.

Insertions.

For an amino acid insertion, the following nomenclature is used:Original amino acid, position, original amino acid, inserted amino acid.Accordingly the insertion of lysine after glycine at position 195 isdesignated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G  G - K -  A

Multiple Alterations.

Variants comprising multiple alterations are separated by addition marks(“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing asubstitution of arginine and glycine at positions 170 and 195 withtyrosine and glutamic acid, respectively.

Different Alterations.

Where different alterations can be introduced at a position, thedifferent alterations are separated by a comma, e.g., “Arg170Tyr,Glu”represents a substitution of arginine at position 170 with tyrosine orglutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates thefollowing variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to alpha-amylase variants comprising analteration at one or more positions corresponding to any of positions 1,2, 68, 71, 126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239,279, 316, 318, 360, 416, 437 and 450 of SEQ ID NO: 1, wherein thevariant has at least 60% and less than 100% sequence identity to (i) themature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQ ID NO: 1, aminoacids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQ ID NO: 3,amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484 of SEQ ID NO:5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485 of SEQ IDNO: 7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to 485 of SEQID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids 1 to 485 ofSEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12, amino acids 1 to483 of SEQ ID NO: 13 or amino acids 1 to 481 of SEQ ID NO: 14, andwherein the variant has alpha-amylase activity.

Preferably, the variants are isolated.

Variants

A variant of the invention comprises an alteration at one or more(several) positions corresponding to any of positions 1, 2, 68, 71, 126,133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318,360, 416, 437 and 450 of SEQ ID NO: 1.

In one embodiment, the variant comprises an insertion or a substitutionat a position corresponding to position 1 with Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,in particular an insertion of or a substitution with Phe, His, Tyr orTrp, preferably His or Trp.

In one embodiment, the variant comprises an insertion or a substitutionat a position corresponding to position 2 with Ala, Arg, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,in particular an insertion of or a substitution with Phe, His, Tyr orTrp, preferably His or Trp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 68 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Phe, Tyr or Trp, preferably with Trp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 71 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Phe, His, Tyr or Trp, preferably with Trp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 126 with Ala, Arg, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Phe, His, Tyr or Trp, preferably with Trp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 133 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Phe, Tyr or Trp, preferably with Tyr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 142 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Phe, Tyr or Trp, preferably with Trp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 144 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr or Val, inparticular with Phe, His, Tyr or Trp, preferably with Trp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 156 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp or Val, inparticular with Phe, His or Trp, preferably with Trp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 158 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp or Val, inparticular with Phe, His or Trp, preferably with Trp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 176 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Leu.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 185 with Ala, Arg, Asn, Asp, Cys, Gln, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Pro.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 201 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Tyr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 205 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Tyr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 213 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Thr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 239 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val, inparticular with Ala or Gin.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 279 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with His, Tyr or Trp, preferably with Trp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 316 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Phe, Tyr or Trp, preferably with Trp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 318 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Phe, His, Tyr or Trp, preferably with Trp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 360 with Ala, Arg, Asn, Asp, Cys, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Ser.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 416 with Ala, Arg, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Val.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 437 with Ala, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Phe, His, Tyr or Trp, preferably with Trp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 450 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Phe, Tyr or Trp, preferably with Trp.

The variant has sequence identity of at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100%, to (i) the mature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQ IDNO: 1, amino acids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQID NO: 3, amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484 ofSEQ ID NO: 5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485of SEQ ID NO: 7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to485 of SEQ ID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids1 to 485 of SEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12, aminoacids 1 to 483 of SEQ ID NO: 13 or amino acids 1 to 481 of SEQ ID NO:14.

In a preferred embodiment, the variant has at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99%, but lessthan 100%, sequence identity to (i) the mature polypeptide of SEQ ID NO:1, or (ii) amino acids 1-483 of SEQ ID NO: 1.

In one embodiment, the variant has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 2,(ii) the mature polypeptide of SEQ ID NO: 2 comprising the deletionsI181*+G182*, (iii) amino acids 1-483 of SEQ ID NO: 2, or (iv) aminoacids 1-483 of SEQ ID NO: 2 comprising the deletions I181*+G182*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 3,(ii) the mature polypeptide of SEQ ID NO: 3 comprising the deletionsT183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 3, or (iv) aminoacids 1-485 of SEQ ID NO: 3 comprising the deletions T183*+G184*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 4,(ii) the mature polypeptide of SEQ ID NO: 4 comprising the deletionsT180*+G181*, (iii) amino acids 1-482 of SEQ ID NO: 4, or (iv) aminoacids 1-482 of SEQ ID NO: 4 comprising the deletions T180*+G181.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 5,(ii) the mature polypeptide of SEQ ID NO: 5 comprising the deletionsT182*+G183*, (iii) amino acids 1-484 of SEQ ID NO: 5, or (iv) aminoacids 1-484 of SEQ ID NO: 5 comprising the deletions T182*+G183*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 6,(ii) the mature polypeptide of SEQ ID NO: 6 comprising the deletionsE178*+G179*, (iii) amino acids 1-483 of SEQ ID NO: 6, or (iv) aminoacids 1-483 of SEQ ID NO: 6 comprising the deletions E178*+G179*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 7,(ii) the mature polypeptide of SEQ ID NO: 7 comprising the deletionsT183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 7, or (iv) aminoacids 1-485 of SEQ ID NO: 7 comprising the deletions T183*+G184*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 8,(ii) the mature polypeptide of SEQ ID NO: 8 comprising the deletionsD183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 8, or (iv) aminoacids 1-485 of SEQ ID NO: 8 comprising the deletions D183*+G184*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 9,(ii) the mature polypeptide of SEQ ID NO: 9 comprising the deletionsD183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 9, or (iv) aminoacids 1-485 of SEQ ID NO: 9 comprising the deletions D183*+G184*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 10,(ii) the mature polypeptide of SEQ ID NO: 10 comprising the deletionsD183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 10, or (iv) aminoacids 1-485 of SEQ ID NO: 10 comprising the deletions D183*+G184*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 11,(ii) the mature polypeptide of SEQ ID NO: 11 comprising the deletionsH183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 11, or (iv) aminoacids 1-485 of SEQ ID NO: 11 comprising the deletions H183*+G184*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 12,or (ii) amino acids 1-480 of SEQ ID NO: 12.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 13,or (ii) amino acids 1-483 of SEQ ID NO: 13.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 14,or (ii) amino acids 1-483 of SEQ ID NO: 14.

A variant of the invention comprises an alteration at one or more (e.g.,several) positions corresponding to any of positions 1, 2, 68, 71, 126,133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318,360, 416, 437 and 450 of SEQ ID NO: 1. In one embodiment, the variantcomprises an alteration at one position corresponding to any ofpositions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176, 185, 201,205, 213, 239, 279, 316, 318, 360, 416, 437 and 450. In anotherembodiment, the variant comprises an alteration at two positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450.In another embodiment, the variant comprises an alteration at threepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416,437 and 450. In another embodiment, the variant comprises an alterationat four positions corresponding to any of positions 1, 2, 68, 71, 126,133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318,360, 416, 437 and 450. In another embodiment, the variant comprises analteration at more than four, e.g., five, six, seven, eight, nine orten, positions corresponding to any of positions 1, 2, 68, 71, 126, 133,142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360,416, 437 and 450. In another embodiment, the variant comprises analteration at more than 10 positions, e.g., 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 positions, corresponding to any of positions 1, 2, 68, 71,126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316,318, 360, 416, 437 and 450.

In an embodiment, the variant comprises an alteration at two or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416,437 and 450. In another embodiment, the variant comprises an alterationat three or more positions corresponding to any of positions 1, 2, 68,71, 126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279,316, 318, 360, 416, 437 and 450. In another embodiment, the variantcomprises an alteration at four or more positions corresponding to anyof positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176, 185, 201,205, 213, 239, 279, 316, 318, 360, 416, 437 and 450. In anotherembodiment, the variant comprises an alteration at five or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416,437 and 450.

In a preferred embodiment, the variant comprises an alteration at two ormore positions corresponding to any of positions 1, 2, 68, 71, 126, 133,142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360,416, 437 and 450, and the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identityto (i) the mature polypeptide of SEQ ID NO: 1, or (ii) amino acids 1-483of SEQ ID NO: 1.

In another preferred embodiment, the variant comprises an alteration attwo or more positions corresponding to any of positions 1, 2, 68, 71,126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316,318, 360, 416, 437 and 450, and the variant has at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In another preferred embodiment, the variant comprises an alteration atthree or more positions corresponding to any of positions 1, 2, 68, 71,126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316,318, 360, 416, 437 and 450, and the variant has at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In another preferred embodiment, the variant comprises an alteration atthree or more positions corresponding to any of positions 1, 2, 68, 71,126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316,318, 360, 416, 437 and 450, and the variant has at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In another preferred embodiment, the variant comprises an alteration atfour or more positions corresponding to any of positions 1, 2, 68, 71,126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316,318, 360, 416, 437 and 450, and the variant has at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In another preferred embodiment, the variant comprises an alteration atfour or more positions corresponding to any of positions 1, 2, 68, 71,126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316,318, 360, 416, 437 and 450, and the variant has at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In another preferred embodiment, the variant comprises an alteration atfive or more positions corresponding to any of positions 1, 2, 68, 71,126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316,318, 360, 416, 437 and 450, and the variant has at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In another preferred embodiment, the variant comprises an alteration atfive or more positions corresponding to any of positions 1, 2, 68, 71,126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316,318, 360, 416, 437 and 450, and the variant has at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant of the invention comprises one or morealterations selected from the group consisting of A1AH, A1AF, A1AY,A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F, N2NY, N2NW, N2H, N2F, N2Y, N2W,H68F, H68Y, H68W, G71F, G71H, G71Y, G71W, N126F, N126H, N126Y, N126W,H133F, H133Y, H133W, H142F, H142Y, H142W, P144F, P144H, P144Y, P144W,Y156F, Y156H, Y156W, Y158F, Y158H, Y158W, K176L, E185P, I201F, I201Y,H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F, H316Y, H316W, L318F,L318H, L318Y, L318W, Q360S, D416V, R437F, R437H, R437Y, R437W, H450F,H450Y and H450W.

In a preferred embodiment, the variant of the invention comprises one ormore substitutions selected from the group consisting of A1H, A1W, N2H,N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V,R437W and H450W.

In another preferred embodiment, the variant comprises two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V,R437W and H450W.

In another embodiment, the variant comprises three or more substitutionsselected from the group consisting of A1H, A1W, N2H, N2W, H68W, G71W,N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W.In another embodiment, the variant comprises four or more substitutionsselected from the group consisting of A1H, A1W, N2H, N2W, H68W, G71W,N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W.

In a preferred embodiment, the variant comprises an alteration at one ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 176, 185, 201, 205, 213, 279, 360, 416 and 437. In anotherpreferred embodiment, the variant comprises an alteration at two or morepositions corresponding to any of positions 2, 68, 133, 142, 156, 158,176, 185, 201, 205, 213, 279, 360, 416 and 437. In another preferredembodiment, the variant comprises an alteration at three or morepositions corresponding to any of positions 2, 68, 133, 142, 156, 158,176, 185, 201, 205, 213, 279, 360, 416 and 437. In another preferredembodiment, the variant comprises an alteration at four or morepositions corresponding to any of positions 2, 68, 133, 142, 156, 158,176, 185, 201, 205, 213, 279, 360, 416 and 437. In another preferredembodiment, the variant comprises an alteration at five or morepositions corresponding to any of positions 2, 68, 133, 142, 156, 158,176, 185, 201, 205, 213, 279, 360, 416 and 437. In another preferredembodiment, the variant comprises an alteration at six or more positionscorresponding to any of positions 2, 68, 133, 142, 156, 158, 176, 185,201, 205, 213, 279, 360, 416 and 437.

In a preferred embodiment, the variant comprises one or moresubstitutions selected from the group consisting of N2H, H68W, H133Y,H142W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, F279W, Q360S,D416V and R437W, and the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identityto (i) the mature polypeptide of SEQ ID NO: 1, or (ii) amino acids 1-483of SEQ ID NO: 1.

In another preferred embodiment, the variant comprises two or moresubstitutions selected from the group consisting of N2H, H68W, H133Y,H142W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, F279W, Q360S,D416V and R437W, and the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identityto (i) the mature polypeptide of SEQ ID NO: 1, or (ii) amino acids 1-483of SEQ ID NO: 1.

In another preferred embodiment, the variant comprises three or moresubstitutions selected from the group consisting of N2H, H68W, H133Y,H142W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, F279W, Q360S,D416V and R437W, and the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identityto (i) the mature polypeptide of SEQ ID NO: 1, or (ii) amino acids 1-483of SEQ ID NO: 1.

In another preferred embodiment, the variant comprises four or moresubstitutions selected from the group consisting of N2H, H68W, H133Y,H142W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, F279W, Q360S,D416V and R437W, and the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identityto (i) the mature polypeptide of SEQ ID NO: 1, or (ii) amino acids 1-483of SEQ ID NO: 1.

In one embodiment, the variant comprises an insertion or a substitutionat a position corresponding to position 1, in particular the insertionA1AH or A1AW or the substitution A1H or A1W, in combination with analteration at one or more positions corresponding to any of positions 2,68, 71, 126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279,316, 318, 360, 416, 437 and 450, in particular one or more alterationsselected from the group consisting of N2NH, N2NW, N2H, N2W, H68W, G71W,N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W.

In a preferred embodiment, the variant comprises an insertion or asubstitution at a position corresponding to position 1, in particularthe insertion A1AH or A1AW or the substitution A1H or A1W, incombination with an alteration at one or more positions corresponding toany of positions 2, 68, 71, 126, 133, 142, 144, 156, 158, 176, 185, 201,205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particular oneor more alterations selected from the group consisting of N2NH, N2NW,N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L,E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W, and the variant has at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii) aminoacids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises an insertion or asubstitution at a position corresponding to position 1, in particularthe insertion A1AH or A1AW or the substitution A1H or A1W, incombination with an alteration at one or more positions corresponding toany of positions 2, 68, 71, 126, 133, 142, 144, 156, 158, 176, 185, 201,205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particular oneor more alterations selected from the group consisting of N2NH, N2NW,N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L,E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W, and the variant has at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity to (i) the mature polypeptide of SEQ ID NO: 14, or (ii) aminoacids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises an insertion or a substitutionat a position corresponding to position 2, in particular the insertionN2NH or N2NW or the substitution N2H or N2W, in combination with analteration at one or more positions corresponding to any of positions 1,68, 71, 126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279,316, 318, 360, 416, 437 and 450, in particular one or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, H68W, G71W,N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W.

In a preferred embodiment, the variant comprises an insertion or asubstitution at a position corresponding to position 2, in particularthe insertion N2NH or N2NW or the substitution N2H or N2W, incombination with an alteration at one or more positions corresponding toany of positions 1, 68, 71, 126, 133, 142, 144, 156, 158, 176, 185, 201,205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particular oneor more alterations selected from the group consisting of A1AH, A1AW,A1H, A1W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L,E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W, and the variant has at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii) aminoacids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises an insertion or asubstitution at a position corresponding to position 2, in particularthe insertion N2NH or N2NW or the substitution N2H or N2W, incombination with an alteration at one or more positions corresponding toany of positions 1, 68, 71, 126, 133, 142, 144, 156, 158, 176, 185, 201,205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particular oneor more alterations selected from the group consisting of A1AH, A1AW,A1H, A1W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L,E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W, and the variant has at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity to (i) the mature polypeptide of SEQ ID NO: 14, or (ii) aminoacids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 68, in particular the substitution H68W, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 71, 126, 133, 142, 144, 156, 158, 176, 185,201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L,E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 68, in particular the substitutionH68W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450,in particular one or more substitutions selected from the groupconsisting of A1H, A1W, N2H, N2W, G71W, N126W, H133Y, H142W, P144W,Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W,H316W, L318W, Q360S, D416V, R437W and H450W, and the variant has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity to (i) the mature polypeptide of SEQ ID NO:1, or (ii) amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 68, in particular the substitutionH68W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450,in particular one or more substitutions selected from the groupconsisting of A1H, A1W, N2H, N2W, G71W, N126W, H133Y, H142W, P144W,Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W,H316W, L318W, Q360S, D416V, R437W and H450W, and the variant has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity to (i) the mature polypeptide of SEQ ID NO:14, or (ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 71, in particular the substitution G71W, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 126, 133, 142, 144, 156, 158, 176, 185,201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L,E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 71, in particular the substitutionG71W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450,in particular one or more substitutions selected from the groupconsisting of A1H, A1W, N2H, N2W, H68W, N126W, H133Y, H142W, P144W,Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W,H316W, L318W, Q360S, D416V, R437W and H450W, and the variant has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity to (i) the mature polypeptide of SEQ ID NO:1, or (ii) amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 71, in particular the substitutionG71W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450,in particular one or more substitutions selected from the groupconsisting of A1H, A1W, N2H, N2W, H68W, N126W, H133Y, H142W, P144W,Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W,H316W, L318W, Q360S, D416V, R437W and H450W, and the variant has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity to (i) the mature polypeptide of SEQ ID NO:14, or (ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 126, in particular the substitution N126W, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 133, 142, 144, 156, 158, 176, 185,201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, H133Y, H142W, P144W, Y156W, Y158W, K176L,E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 126, in particular the substitutionN126W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 133, 142, 144, 156, 158,176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, H133Y, H142W, P144W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 126, in particular the substitutionN126W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 133, 142, 144, 156, 158,176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, H133Y, H142W, P144W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 133, in particular the substitution H133Y, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 142, 144, 156, 158, 176, 185,201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H142W, P144W, Y156W, Y158W, K176L,E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 133, in particular the substitutionH133Y, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 142, 144, 156, 158,176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H142W, P144W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 133, in particular the substitutionH133Y, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 142, 144, 156, 158,176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H142W, P144W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 142, in particular the substitution H142W, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 144, 156, 158, 176, 185,201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, P144W, Y156W, Y158W, K176L,E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 142, in particular the substitutionH142W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 144, 156, 158,176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, P144W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 142, in particular the substitutionH142W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 144, 156, 158,176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, P144W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 144, in particular the substitution P144W, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 142, 156, 158, 176, 185,201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, Y156W, Y158W, K176L,E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 144, in particular the substitutionP144W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 156, 158,176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 144, in particular the substitutionP144W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 156, 158,176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 156, in particular the substitution Y156W, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 142, 144, 158, 176, 185,201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y158W, K176L,E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 156, in particular the substitutionY156W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 158,176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 156, in particular the substitutionY156W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 158,176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 158, in particular the substitution Y158W, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 176, 185,201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, K176L,E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 158, in particular the substitutionY158W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 158, in particular the substitutionY158W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 176, in particular the substitution K176L, incombination with an alteration at one or more positions corresponding toany of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 185, 201,205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particular oneor more alterations selected from the group consisting of A1AH, A1AW,A1H, A1W, N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W,Y156W, Y158W, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 176, in particular the substitutionK176L, in combination with an alteration at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more alterations selected from the group consisting ofA1AH, A1AW, A1H, A1W, N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y,H142W, P144W, Y156W, Y158W, E185P, I201Y, H205Y, K213T, S239A, S239Q,F279W, H316W, L318W, Q360S, D416V, R437W and H450W, and the variant hasat least 60%, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% sequence identity to (i) the mature polypeptide ofSEQ ID NO: 1, or (ii) amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 176, in particular the substitutionK176L, in combination with an alteration at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more alterations selected from the group consisting ofA1AH, A1AW, A1H, A1W, N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y,H142W, P144W, Y156W, Y158W, E185P, I201Y, H205Y, K213T, S239A, S239Q,F279W, H316W, L318W, Q360S, D416V, R437W and H450W, and the variant hasat least 60%, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% sequence identity to (i) the mature polypeptide ofSEQ ID NO: 14, or (ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 185, in particular the substitution E185P, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176,201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W,K176L, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 185, in particular the substitutionE185P, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 185, in particular the substitutionE185P, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 201, in particular the substitution I201Y, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176,185, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W,K176L, E185P, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 201, in particular the substitutionI201Y, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 201, in particular the substitutionI201Y, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 205, in particular the substitution H205Y, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176,185, 201, 213, 239, 279, 316, 318, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W,K176L, E185P, I201Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 205, in particular the substitutionH205Y, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 205, in particular the substitutionH205Y, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 213, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 213, in particular the substitution K213T, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176,185, 201, 205, 239, 279, 316, 318, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, S239A, S239Q, F279W, H316W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 213, in particular the substitutionK213T, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 213, in particular the substitutionK213T, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 239, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 239, in particular the substitution S239A orS239Q, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 279, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, F279W, H316W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 239, in particular the substitutionS239A or S239Q, in combination with a substitution at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 279, 316, 318, 360, 416, 437 and450, in particular one or more substitutions selected from the groupconsisting of A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W,P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, F279W, H316W,L318W, Q360S, D416V, R437W and H450W, and the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or(ii) amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 239, in particular the substitutionS239A or S239Q, in combination with a substitution at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 279, 316, 318, 360, 416, 437 and450, in particular one or more substitutions selected from the groupconsisting of A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W,P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, F279W, H316W,L318W, Q360S, D416V, R437W and H450W, and the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 279, in particular the substitution F279W, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176,185, 201, 205, 213, 239, 316, 318, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, H316W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 279, in particular the substitutionF279W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 279, in particular the substitutionF279W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 316, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, H316W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 316, in particular the substitution H316W, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176,185, 201, 205, 213, 239, 279, 318, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, L318W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 316, in particular the substitutionH316W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 316, in particular the substitutionH316W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 318, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, L318W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 318, in particular the substitution L318W, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176,185, 201, 205, 213, 239, 279, 316, 360, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, Q360S,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 318, in particular the substitutionL318W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 318, in particular the substitutionL318W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 360, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,Q360S, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 360, in particular the substitution Q360S, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176,185, 201, 205, 213, 239, 279, 316, 318, 416, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,D416V, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 360, in particular the substitutionQ360S, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 360, in particular the substitutionQ360S, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 416, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, D416V, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 416, in particular the substitution D416V, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176,185, 201, 205, 213, 239, 279, 316, 318, 360, 437 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, R437W and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 416, in particular the substitutionD416V, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 416, in particular the substitutionD416V, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 437 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, R437W and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 437, in particular the substitution R437W, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176,185, 201, 205, 213, 239, 279, 316, 318, 360, 416 and 450, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V and H450W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 437, in particular the substitutionR437W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, D416V and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 437, in particular the substitutionR437W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416 and 450, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, D416V and H450W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 450, in particular the substitution H450W, incombination with a substitution at one or more positions correspondingto any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176,185, 201, 205, 213, 239, 279, 316, 318, 360, 416 and 437, in particularone or more substitutions selected from the group consisting of A1H,A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W,K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V and R437W.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 450, in particular the substitutionH450W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416 and 437, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, D416V and R437W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii)amino acids 1-483 of SEQ ID NO: 1.

In a preferred embodiment, the variant comprises a substitution at aposition corresponding to position 450, in particular the substitutionH450W, in combination with a substitution at one or more positionscorresponding to any of positions 1, 2, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416 and 437, inparticular one or more substitutions selected from the group consistingof A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, D416V and R437W, and the variant has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%sequence identity to (i) the mature polypeptide of SEQ ID NO: 14, or(ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the variant further comprises a substitution at oneor more positions, e.g., at two or more positions or at three or morepositions, corresponding to positions 15, 48, 49, 107, 138, 156, 181,187, 188, 190, 197, 209, 239, 255, 264, 299, 474 and 475 of SEQ ID NO:1.

In a preferred embodiment, the variant further comprises a substitutionat one or more positions, e.g., at two or more positions or at three ormore positions, corresponding to positions 15, 48, 49, 107, 138, 156,181, 187, 188, 190, 197, 209, 239, 255, 264, 299, 474 and 475 of SEQ IDNO: 1, and the variant has at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98% or at least 99% sequence identity to (i)the mature polypeptide of SEQ ID NO: 1, or (ii) amino acids 1-483 of SEQID NO: 1.

In a preferred embodiment, the variant further comprises a substitutionat one or more positions, e.g., at two or more positions or at three ormore positions, corresponding to positions 15, 48, 49, 107, 138, 156,181, 187, 188, 190, 197, 209, 239, 255, 264, 299, 474 and 475 of SEQ IDNO: 1, and the variant has at least 60%, e.g., at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98% or at least 99% sequence identity to (i)the mature polypeptide of SEQ ID NO: 14, or (ii) amino acids 1-481 ofSEQ ID NO: 14.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 15 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Leu, Ser or Thr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 48 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Ala.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 49 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val, inparticular with Gly, His, Ile or Leu.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 107 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Ala.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 138 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr or Val, inparticular with Phe or Tyr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 156 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Tyr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 181 with Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Asp, Glu or Thr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 187 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val, inparticular with Asp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 188 with Ala, Arg, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Ser or Thr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 190 with Ala, Arg, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Phe.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 197 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Ile, Leu, Ser, Thr or Val.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 209 with Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Val.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 239 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val, inparticular with Ala, Asn, Asp, Gln, Glu or Met.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 255 with Ala, Arg, Asn, Asp, Cys, Gln, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Gly or Pro.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 264 with Ala, Arg, Asn, Asp, Cys, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Ser.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 299 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Arg.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 474 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Arg, Gln, Glu or Lys.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 475 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Arg, Gln, Glu or Lys.

In a preferred embodiment, the variant comprises one or more, e.g., twoor more, or three or more, substitutions selected from the groupconsisting of M15T, M15S, M15L, G48A, T49L, T49I, G107A, W138Y, W138F,H156Y, A181T, A181E, A181D, S187D, N188S, N188T, N190F, M197S, M197T,M197V, M197L, M1971, A209V, S239Q, S239E, S239N, S239D, S239A, S239M,E255P, E255G, Q264S, G299R, G474K, G474R, G474E, G474Q, G475K, G475R,G475E and G475Q.

In another preferred embodiment, the variant comprises one or more,e.g., two or more, or three or more, substitutions selected from thegroup consisting of M15T, M15S, M15L, G48A, T49L, T49I, G107A, W138Y,W138F, H156Y, A181T, A181E, A181D, S187D, N188S, N188T, N190F, M197S,M197T, M197V, M197L, M1971, A209V, S239Q, S239E, S239N, S239D, S239A,S239M, E255P, E255G, Q264S, G299R, G474K, G474R, G474E, G474Q, G475K,G475R, G475E and G475Q, and the variant has at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity to (i) the mature polypeptide of SEQ ID NO: 1, or (ii) aminoacids 1-483 of SEQ ID NO: 1.

In another preferred embodiment, the variant comprises one or more,e.g., two or more, or three or more, substitutions selected from thegroup consisting of M15T, M15S, M15L, G48A, T49L, T49I, G107A, W138Y,W138F, H156Y, A181T, A181E, A181D, S187D, N188S, N188T, N190F, M197S,M197T, M197V, M197L, M1971, A209V, S239Q, S239E, S239N, S239D, S239A,S239M, E255P, E255G, Q264S, G299R, G474K, G474R, G474E, G474Q, G475K,G475R, G475E and G475Q, and the variant has at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequenceidentity to (i) the mature polypeptide of SEQ ID NO: 14, or (ii) aminoacids 1-481 of SEQ ID NO: 14.

In a preferred embodiment, a variant of the invention comprises a set ofsubstitutions selected from the group consisting of:

A1H+N2W+K176L+E185P,

A1W+N2H+K176L+E185P,

N2H+H68W+H133Y+K176L+E185P,

N2H+H68W+Y156W+K176L+E185P,

N2H+H68W+Y158W+K176L+E185P,

N2H+H68W+K176L+E185P,

N2H+H68W+K176L+E185P+I201Y+H205Y+D207V+V209D,

N2H+H68W+K176L+E185P+F279W,

N2H+H133Y+K176L+E185P+H316W+R437W,

N2H+H133Y+K176L+E185P+Q360S+R437W,

N2H+H142W+K176L+E185P+H316W+R437W,

N2H+H142W+K176L+E185P+Q360S+R437W,

N2H+P144W+K176L+E185P,

N2H+Y156W+Y158W+K176L+E185P+H316W+R437W,

N2H+Y156W+K176L+E185P+Q360S+R437W,

N2H+Y158W+K176L+E185P+I201Y+H205Y+D207V+V209D+H316W,

N2H+K176L+E185P,

N2H+K176L+E185P+H316W,

N2H+K176L+E185P+H316W+L318W+R437W,

N2H+K176L+E185P+H316W+Q360S+R437W,

N2H+K176L+E185P+H316W+R437W,

N2H+K176L+E185P+R437W,

N2H+K176L+E185P+Q360S+R437W,

N2H+K176L+E185P+H316W+Q360S+R437W,

N2H+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H68W+K176L+E185P,

H68W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H68W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

G71W+K176L+E185P,

N126W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H133Y+Y158W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H133Y+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H142W+Y158W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H142W+K176L+E185P,

H142W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H142W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

P144W+K176L+E185P,

Y156W+Y158W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

Y156W+Y158W+K176L+E185P+H316W+R437W,

Y156W+K176L+E185P+Q360S+R437W,

Y156W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

Y158W+K176L+E185P,

Y158W+K176L+E185P+I201Y+H205Y+D207V+V209D+H316W,

Y158W+K176L+E185P+I201Y+H205Y+K213T+H316L+L318W+Q360S+D416V+R437W,

Y158W+K176L+E185P+I201Y+H205Y+K213T+H316W+Q360S+D416V+R437W,

Y158W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

Y158W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

K176L+E185P,

K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

K176L+E185P+I201Y+H205Y+R437W,

K176L+E185P+F279W,

K176L+E185P+H316W,

K176L+E185P+L318W,

K176L+E185P+H450W,

K176L+I201Y+H205Y+K213T+S239Q+Q360S+D416V+R437W,

K176L+I201Y+H205Y+K213T+H316W+Q360S+D416V+R437W,

K176L+I201Y+H205Y+K213T+L318W+Q360S+D416V+R437W,

K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

K176L+I201Y+H205Y+K213T+Q360S+R437W,

K176L+I201Y+H205Y+K213T+D416V+R437W, and

K176L+I201Y+H205Y+Q360S+D416V+R437W.

In another preferred embodiment, a variant of the invention comprises aset of substitutions selected from the group consisting of:

A1H+N2W+K176L+E185P,

A1W+N2H+K176L+E185P,

N2H+H68W+H133Y+K176L+E185P,

N2H+H68W+Y156W+K176L+E185P,

N2H+H68W+Y158W+K176L+E185P,

N2H+H68W+K176L+E185P,

N2H+H68W+K176L+E185P+I201Y+H205Y+D207V+V209D,

N2H+H68W+K176L+E185P+F279W,

N2H+H133Y+K176L+E185P+H316W+R437W,

N2H+H133Y+K176L+E185P+Q360S+R437W,

N2H+H142W+K176L+E185P+H316W+R437W,

N2H+H142W+K176L+E185P+Q360S+R437W,

N2H+P144W+K176L+E185P,

N2H+Y156W+Y158W+K176L+E185P+H316W+R437W,

N2H+Y156W+K176L+E185P+Q360S+R437W,

N2H+Y158W+K176L+E185P+I201Y+H205Y+D207V+V209D+H316W,

N2H+K176L+E185P,

N2H+K176L+E185P+H316W,

N2H+K176L+E185P+H316W+L318W+R437W,

N2H+K176L+E185P+H316W+Q360S+R437W,

N2H+K176L+E185P+H316W+R437W,

N2H+K176L+E185P+R437W,

N2H+K176L+E185P+Q360S+R437W,

N2H+K176L+E185P+H316W+Q360S+R437W,

N2H+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H68W+K176L+E185P,

H68W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H68W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

G71W+K176L+E185P,

N126W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H133Y+Y158W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H133Y+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H142W+Y158W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H142W+K176L+E185P,

H142W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H142W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

P144W+K176L+E185P,

Y156W+Y158W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

Y156W+Y158W+K176L+E185P+H316W+R437W,

Y156W+K176L+E185P+Q360S+R437W,

Y156W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

Y158W+K176L+E185P,

Y158W+K176L+E185P+I201Y+H205Y+D207V+V209D+H316W,

Y158W+K176L+E185P+I201Y+H205Y+K213T+H316L+L318W+Q360S+D416V+R437W,

Y158W+K176L+E185P+I201Y+H205Y+K213T+H316W+Q360S+D416V+R437W,

Y158W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

Y158W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

K176L+E185P,

K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

K176L+E185P+I201Y+H205Y+R437W,

K176L+E185P+F279W,

K176L+E185P+H316W,

K176L+E185P+L318W,

K176L+E185P+H450W,

K176L+I201Y+H205Y+K213T+S239Q+Q360S+D416V+R437W,

K176L+I201Y+H205Y+K213T+H316W+Q360S+D416V+R437W,

K176L+I201Y+H205Y+K213T+L318W+Q360S+D416V+R437W,

K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

K176L+I201Y+H205Y+K213T+Q360S+R437W,

K176L+I201Y+H205Y+K213T+D416V+R437W, and

K176L+I201Y+H205Y+Q360S+D416V+R437W.

and the variant has at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% sequence identity to (i) themature polypeptide of SEQ ID NO: 14, or (ii) amino acids 1-481 of SEQ IDNO: 14.

In one embodiment, the variant further comprises a deletion at both ofthe two positions immediately before the position corresponding toposition 180 of SEQ ID NO: 1. I.e., a deletion of the two amino acidscorresponding to positions 181 and 182 of SEQ ID NO: 2.

In another embodiment, the variant further comprises a deletion of twoamino acids after the position corresponding to position 177 of SEQ IDNO: 1 and before the position corresponding to position 180 of SEQ IDNO: 1. I.e., a deletion of two amino acids in the R179-G180-I181-G182peptide of SEQ ID NO: 2, or homologous amino acids in any of SEQ ID NO:3 to 11.

The variants may further comprise one or more (e.g., several) additionalalterations, e.g., one or more (e.g., several) additional substitutions.

The additional amino acid changes may be of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of 1-30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for alpha-amylase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

The variants may consist of 300 to 700, e.g., 350 to 650, 400 to 600,450 to 500 or 470 to 490, amino acids.

In a preferred embodiment, the variant has increased thermostabilitycompared to the mature polypeptide of SEQ ID NO: I at high temperature,low calcium and low pH in the presence of at least 0.1% starch, e.g., inthe presence of 0.9% or 1% starch.

In the context of the present invention “high temperature” meanstemperatures from 70-120° C., preferably 80-100° C., more preferably85-95° C.

In the context of the present invention the term “low pH” means a pH inthe range from 4-6, preferably 4.2-5.5, more preferably 4.5-5.

In the context of the present invention the term “low calcium” meansfree calcium levels lower than 60 ppm, preferably 40 ppm, morepreferably 25 ppm, even more preferably 5 ppm calcium. 0.125 mM CaCl₂provides 5 ppm calcium.

In one embodiment, the variant has increased thermostability compared tothe mature polypeptide of SEQ ID NO: 1 when incubated with 0.125 mMCaCl₂, 0.1% starch at 95° C. and pH 4.8. In another embodiment, thevariant has increased thermostability compared to the mature polypeptideof SEQ ID NO: 1 when incubated with 0.125 mM CaCl₂, 0.1% starch at 95°C. and pH 4.5.

In another embodiment, the variant has increased thermostabilitycompared to the mature polypeptide of SEQ ID NO: 1 when incubated with0.125 mM CaCl₂, 0.9% starch at 95° C. and pH 4.8. In another embodiment,the variant has increased thermostability compared to the maturepolypeptide of SEQ ID NO: 1 when incubated with 0.125 mM CaCl₂, 0.9%starch at 95° C. and pH 4.5.

In another embodiment, the variant has increased thermostabilitycompared to the mature polypeptide of SEQ ID NO: 1 when incubated with0.125 mM CaCl₂, 1% starch at 95° C. and pH 4.8.

In another embodiment, the variant has increased thermostabilitycompared to the mature polypeptide of SEQ ID NO: 1 when incubated with0.125 mM CaCl₂, 1% starch at 95° C. and pH 4.5.

The skilled person will know how to determine thermostability of theenzymes. It may be done by determining the residual activity half-lifeas shown in the Examples of the present application. The skilled personwill know how to choose the relevant conditions for the assay, such asthe incubation time.

In a preferred embodiment, the variant has an increased residualactivity half-life compared to the mature polypeptide of SEQ ID NO: 1 athigh temperature, low calcium and low pH in the presence of at least0.1% starch, e.g., in the presence of 0.9% or 1% starch. In onepreferred embodiment, the variant has an increased residual activityhalf-life compared to the mature polypeptide of SEQ ID NO: 1 whenincubated with 0.125 mM CaCl₂, 0.1% starch at 95° C. and pH 4.8. Inanother preferred embodiment, the variant has an increased residualactivity half-life compared to the mature polypeptide of SEQ ID NO: 1when incubated with 0.125 mM CaCl₂, 0.1% starch at 95° C. and pH 4.5. Inanother preferred embodiment, the variant has an increased residualactivity half-life compared to the mature polypeptide of SEQ ID NO: 1when incubated with 0.125 mM CaCl₂, 0.9% starch at 95° C. and pH 4.8. Inanother preferred embodiment, the variant has an increased residualactivity half-life compared to the mature polypeptide of SEQ ID NO: 1when incubated with 0.125 mM CaCl₂, 0.9% starch at 95° C. and pH 4.5. Inanother preferred embodiment, the variant has an increased residualactivity half-life compared to the mature polypeptide of SEQ ID NO: 1when incubated with 0.125 mM CaCl₂, 1% starch at 95° C. and pH 4.8. Inanother preferred embodiment, the variant has an increased residualactivity half-life compared to the mature polypeptide of SEQ ID NO: 1when incubated with 0.125 mM CaCl₂, 1% starch at 95° C. and pH 4.5.

In a preferred embodiment, the variant has a residual activityhalf-life, when incubated with 0.125 mM CaCl₂, 0.9% starch at 95° C. andpH 4.8, which is at least 5 minutes, e.g., at least 7 minutes or atleast 10 minutes. In a more preferred embodiment, the variant has aresidual activity half-life, when incubated with 0.125 mM CaCl₂, 0.9%starch at 95° C. and pH 4.8, which is at least 15 minutes, e.g., atleast 20 minutes or at least 30 minutes. In one embodiment, the varianthas a residual activity half-life, when incubated with 0.125 mM CaCl₂,0.9% starch at 95° C. and pH 4.8, which is at least 40 minutes, e.g., atleast 50 minutes or at least 60 minutes.

In a preferred embodiment, the variant has a residual activityhalf-life, when incubated with 0.125 mM CaCl₂, 0.9% starch at 95° C. andpH 4.5, which is at least 1 minute, e.g., at least 2 minutes or at least5 minutes. In a more preferred embodiment, the variant has a residualactivity half-life, when incubated with 0.125 mM CaCl₂, 0.9% starch at95° C. and pH 4.5, which is at least 8 minutes, e.g., at least 10minutes or at least 12 minutes. In one embodiment, the variant has aresidual activity half-life, when incubated with 0.125 mM CaCl₂, 0.9%starch at 95° C. and pH 4.8, which is at least 15 minutes, e.g., atleast 20 minutes or at least 25 minutes.

In a preferred embodiment, the variant has increased thermostabilitycompared to the parent enzyme at high temperature, low calcium and lowpH in the presence of at least 0.1% starch, e.g., in the presence of0.9% or 1% starch.

In another preferred embodiment, the variant has an increased residualactivity half-life compared to the parent enzyme at high temperature,low calcium and low pH in the presence of at least 0.1% starch, e.g., inthe presence of 0.9% or 1% starch.

In one embodiment, the variant has increased thermostability, e.g., anincreased residual activity half-life, compared to the parent enzymewhen incubated with 0.125 mM CaCl₂, 0.1% starch at 95° C. and pH 4.8. Inanother embodiment, the variant has increased thermostability, e.g., anincreased residual activity half-life, compared to the parent enzymewhen incubated with 0.125 mM CaCl₂, 0.1% starch at 95° C. and pH 4.5.

In one embodiment, the variant has increased thermostability, e.g., anincreased residual activity half-life, compared to the parent enzymewhen incubated with 0.125 mM CaCl₂, 0.9% starch at 95° C. and pH 4.8. Inanother embodiment, the variant has increased thermostability, e.g., anincreased residual activity half-life, compared to the parent enzymewhen incubated with 0.125 mM CaCl₂, 0.9% starch at 95° C. and pH 4.5.

In one embodiment, the variant has increased thermostability, e.g., anincreased residual activity half-life, compared to the parent enzymewhen incubated with 0.125 mM CaCl₂, 1% starch at 95° C. and pH 4.8. Inanother embodiment, the variant has increased thermostability, e.g., anincreased residual activity half-life, compared to the parent enzymewhen incubated with 0.125 mM CaCl₂, 1% starch at 95° C. and pH 4.5.

In other embodiments, the variant has improved catalytic efficiency,improved catalytic rate, improved chemical stability, improved oxidationstability, improved specific activity, improved stability under storageconditions, improved substrate binding, improved substrate cleavage,improved substrate specificity, improved substrate stability, improvedsurface properties, improved thermal activity, or improvedthermostability compared to the parent enzyme.

Parent Alpha-Amylase

The variant is preferably a variant of a parent alpha-amylase selectedfrom the group consisting of:

(a) a polypeptide having at least 60% sequence identity to (i) themature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQ ID NO: 1, aminoacids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQ ID NO: 3,amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484 of SEQ ID NO:5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485 of SEQ IDNO: 7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to 485 of SEQID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids 1 to 485 ofSEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12, amino acids 1 to483 of SEQ ID NO: 13 or amino acids 1 to 481 of SEQ ID NO: 14; or(b) a fragment of the mature polypeptide of any of SEQ ID NOs: 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, which has alpha-amylaseactivity.

In one embodiment, the parent alpha-amylase has at least 65%, e.g., atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of any of SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) aminoacids 1 to 483 of SEQ ID NO: 1, amino acids 1 to 483 of SEQ ID NO: 2,amino acids 1 to 485 of SEQ ID NO: 3, amino acids 1 to 482 of SEQ ID NO:4, amino acids 1 to 484 of SEQ ID NO: 5, amino acids 1 to 483 of SEQ IDNO: 6, amino acids 1 to 485 of SEQ ID NO: 7, amino acids 1 to 485 of SEQID NO: 8, amino acids 1 to 485 of SEQ ID NO: 9, amino acids 1 to 485 ofSEQ ID NO: 10, amino acids 1 to 485 of SEQ ID NO: 11, amino acids 1 to480 of SEQ ID NO: 12, amino acids 1 to 483 of SEQ ID NO: 13 or aminoacids 1 to 481 of SEQ ID NO: 14.

In one embodiment, the parent alpha-amylase has at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 1, or (ii) amino acids 1-483 of SEQ ID NO: 1.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 2,(ii) the mature polypeptide of SEQ ID NO: 2 comprising the deletionsI181*+G182*, (iii) amino acids 1-483 of SEQ ID NO: 2, or (iv) aminoacids 1-483 of SEQ ID NO: 2 comprising the deletions I181+G182*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 3,(ii) the mature polypeptide of SEQ ID NO: 3 comprising the deletionsT183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 3, or (iv) aminoacids 1-485 of SEQ ID NO: 3 comprising the deletions T183*+G184*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 4,(ii) the mature polypeptide of SEQ ID NO: 4 comprising the deletionsT180*+G181*, (iii) amino acids 1-482 of SEQ ID NO: 4, or (iv) aminoacids 1-482 of SEQ ID NO: 4 comprising the deletions T180*+G181*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 5,(ii) the mature polypeptide of SEQ ID NO: 5 comprising the deletionsT182*+G183*, (iii) amino acids 1-484 of SEQ ID NO: 5, or (iv) aminoacids 1-484 of SEQ ID NO: 5 comprising the deletions T182*+G183*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 6,(ii) the mature polypeptide of SEQ ID NO: 6 comprising the deletionsE178*+G179*, (iii) amino acids 1-483 of SEQ ID NO: 6, or (iv) aminoacids 1-483 of SEQ ID NO: 6 comprising the deletions E178*+G179*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 7,(ii) the mature polypeptide of SEQ ID NO: 7 comprising the deletionsT183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 7, or (iv) aminoacids 1-485 of SEQ ID NO: 7 comprising the deletions T183*+G184*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 8,(ii) the mature polypeptide of SEQ ID NO: 8 comprising the deletionsD183*+G184*, (iii) amino acids 1-486 of SEQ ID NO: 8, or (iv) aminoacids 1-486 of SEQ ID NO: 8 comprising the deletions D183*+G184*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 9,(ii) the mature polypeptide of SEQ ID NO: 9 comprising the deletionsD183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 9, or (iv) aminoacids 1-485 of SEQ ID NO: 9 comprising the deletions D183*+G184*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 10,(ii) the mature polypeptide of SEQ ID NO: 10 comprising the deletionsD183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 10, or (iv) aminoacids 1-485 of SEQ ID NO: 10 comprising the deletions D183*+G184*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 11,(ii) the mature polypeptide of SEQ ID NO: 11 comprising the deletionsH183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 11, or (iv) aminoacids 1-485 of SEQ ID NO: 11 comprising the deletions H183*+G184*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 12,or (ii) amino acids 1-480 of SEQ ID NO: 12.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 13,or (ii) amino acids 1-483 of SEQ ID NO: 13.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 14,or (ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the parent alpha-amylase comprises or consists of themature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14.

In one embodiment, the parent alpha-amylase comprises or consists ofamino acids 1 to 483 of SEQ ID NO: 1, amino acids 1 to 483 of SEQ ID NO:2, amino acids 1 to 485 of SEQ ID NO: 3, amino acids 1 to 482 of SEQ IDNO: 4, amino acids 1 to 484 of SEQ ID NO: 5, amino acids 1 to 483 of SEQID NO: 6, amino acids 1 to 485 of SEQ ID NO: 7, amino acids 1 to 485 ofSEQ ID NO: 8, amino acids 1 to 485 of SEQ ID NO: 9, amino acids 1 to 485of SEQ ID NO: 10, amino acids 1 to 485 of SEQ ID NO: 11, amino acids 1to 480 of SEQ ID NO: 12, amino acids 1 to 483 of SEQ ID NO: 13 or aminoacids 1 to 481 of SEQ ID NO: 14.

In another embodiment, the parent alpha-amylase is a fragment of themature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14, wherein the fragment has alpha-amylase activity.

In one embodiment, the variant has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the parent alpha-amylase.

In one embodiment, the total number of substitutions in the variants ofthe present invention is 2-20, e.g., 2-10 and 2-5, such as 2, 3, 4, 5,6, 7, 8, 9 or 10 substitutions.

The parent may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial alpha-amylase. For example, the parent maybe a Gram-positive bacterial polypeptide such as a Bacillus,Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus,Oceanobacillus, Staphylococcus, Streptococcus, or Streptomycesalpha-amylase, or a Gram-negative bacterial polypeptide such as aCampylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasmaalpha-amylase.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis alpha-amylase.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining a varianthaving alpha-amylase activity, comprising: (a) introducing into a parentalpha-amylase an alteration at one or more positions corresponding toany of positions 1, 2, 68, 71, 126, 133, 142, 144, 156, 158, 176, 185,201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450 of SEQ ID NO:1; and (b) recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga variant of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis crylIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoniglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis crylIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(omithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobactenum, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thome, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series Nov. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Nat. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants. For example, an enzyme assay may be used todetermine the activity of the variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Compositions

The present invention also relates to compositions comprising analpha-amylase variant and at least one additional enzyme. The additionalenzyme(s) may be selected from the group consisting of beta-amylase,cellulase (beta-glucosidase, cellobiohydrolase and endoglucanase),glucoamylase, hemicellulsae (e.g., xylanase), isoamylase, isomerase,lipase, phytase, protease, pullulanase, and/or other enzymes useful in acommercial process in conjunction with an alpha-amylase. The additionalenzyme may also be a second alpha-amylase. Such enzymes are known in theart in starch processing, sugar conversion, fermentations for alcoholand other useful end-products, commercial detergents and cleaning aids,stain removal, fabric treatment or desizing, and the like.

Uses

The variants of the present invention possess valuable propertiesallowing for a variety of industrial applications. In particular, thevariants may be used in detergents, in particular laundry detergentcompositions and dishwashing detergent compositions, hard surfacecleaning compositions, and for desizing textiles, fabrics or garments,production of pulp and paper, beer making, ethanol production, andstarch conversion processes.

The alpha-amylase variants may be used for starch processes, inparticular starch conversion, especially liquefaction of starch (see,e.g., U.S. Pat. No. 3,912,590, EP 252730 and EP 063909, WO 99/19467, andWO 96/28567, which are all hereby incorporated by reference). Alsocontemplated are compositions for starch conversion purposes, which maybeside the variant of the invention also comprise an AMG, pullulanase,and other alpha-amylases.

Further, the variants are particularly useful in the production ofsweeteners and ethanol (see, e.g., U.S. Pat. No. 5,231,017, which ishereby incorporated by reference), such as fuel, drinking and industrialethanol, from starch or whole grains.

The variants may also be used for desizing of textiles, fabrics, andgarments (see, e.g., WO 95/21247, U.S. Pat. No. 4,643,736, and EP119920, which are incorporated herein by reference), beer making orbrewing, and in pulp and paper production or related processes.

Starch Processing

Native starch consists of microscopic granules, which are insoluble inwater at room temperature. When an aqueous starch slurry is heated, thegranules swell and eventually burst, dispersing the starch moleculesinto the solution. During this “gelatinization” process there is adramatic increase in viscosity. As the solids level is 30-40% in atypical industrial process, the starch has to be thinned or “liquefied”so that it can be suitably processed. This reduction in viscosity isprimarily attained by enzymatic degradation in current commercialpractice.

Conventional starch-conversion processes, such as liquefaction andsaccharification processes are described, e.g., in U.S. Pat. No.3,912,590, EP 252730 and EP 063909, which are incorporated herein byreference.

In an embodiment, the conversion process degrading starch to lowermolecular weight carbohydrate components such as sugars or fat replacersincludes a debranching step.

In the case of converting starch into a sugar, the starch isdepolymerized. Such a depolymerization process consists of, e.g., apre-treatment step and two or three consecutive process steps, i.e., aliquefaction process, a saccharification process, and depending on thedesired end-product, an optional isomerization process.

When the desired final sugar product is, e.g., high fructose syrup thedextrose syrup may be converted into fructose. After thesaccharification process, the pH is increased to a value in the range of6-8, e.g., pH 7.5, and the calcium is removed by ion exchange. Thedextrose syrup is then converted into high fructose syrup using, e.g.,an immobilized glucose isomerase.

Production of Fermentation Products

In general, alcohol production (ethanol) from whole grain can beseparated into 4 main steps: milling, liquefaction, saccharification,and fermentation.

The grain is milled in order to open up the structure and allow forfurther processing. Two processes used are wet or dry milling. In drymilling, the whole kernel is milled and used in the remaining part ofthe process. Wet milling gives a very good separation of germ and meal(starch granules and protein) and is with a few exceptions applied atlocations where there is a parallel production of syrups.

In the liquefaction process the starch granules are solubilized byhydrolysis to maltodextrins mostly of a DP higher than 4. The hydrolysismay be carried out by acid treatment or enzymatically by analpha-amylase. Acid hydrolysis is used on a limited basis. The rawmaterial can be milled whole grain or a side stream from starchprocessing.

During a typical enzymatic liquefaction, the long-chained starch isdegraded into branched and linear shorter units (maltodextrins) by analpha-amylase. Enzymatic liquefaction is generally carried out as athree-step hot slurry process. The slurry is heated to between 60-95° C.(e.g., 77-86° C., 80-85° C., or 83-85° C.) and the enzyme(s) is (are)added. The liquefaction process is carried out at 85° C. for 1-2 hours.The pH is generally between 5.5 and 6.2. In order to ensure optimalenzyme stability under these conditions, 1 mM of calcium is added (toprovide about 40 ppm free calcium ions). After such treatment, theliquefied starch will have a “dextrose equivalent” (DE) of 10-15.

The slurry is subsequently jet-cooked at between 95-140° C., e.g.,105-125° C., cooled to 60-95° C. and more enzyme(s) is (are) added toobtain the final hydrolysis. The liquefaction process is carried out atpH 4.5-6.5, typically at a pH between 5 and 6. Milled and liquefiedgrain is also known as mash.

Liquefied starch-containing material is saccharified in the presence ofsaccharifying enzymes such as glucoamylases. The saccharificationprocess may last for 12 hours to 120 hours (e.g., 12 to 90 hours, 12 to60 hours and 12 to 48 hours).

However, it is common to perform a pre-saccharification step for about30 minutes to 2 hours (e.g., 30 to 90 minutes) at a temperature of 30 to65° C., typically around 60° C. which is followed by a completesaccharification during fermentation referred to as simultaneoussaccharification and fermentation (SSF). The pH is usually between4.2-4.8, e.g., pH 4.5. In a simultaneous saccharification andfermentation (SSF) process, there is no holding stage forsaccharification, rather, the yeast and enzymes are added together.

In a typical saccharification process, maltodextrins produced duringliquefaction are converted into dextrose by adding a glucoamylase and adebranching enzyme, such as an isoamylase (U.S. Pat. No. 4,335,208) or apullulanase. The temperature is lowered to 60° C., prior to the additionof the glucoamylase and debranching enzyme. The saccharification processproceeds for 24-72 hours.

Prior to addition of the saccharifying enzymes, the pH is reduced tobelow 4.5, while maintaining a high temperature (above 95° C.), toinactivate the liquefying alpha-amylase. This process reduces theformation of short oligosaccharide called “panose precursors,” whichcannot be hydrolyzed properly by the debranching enzyme. Normally, about0.2-0.5% of the saccharification product is the branched trisaccharidepanose (Glc pα1-6Glc pα1-4Glc), which cannot be degraded by apullulanase. If active amylase from the liquefaction remains presentduring saccharification (i.e., no denaturing), the amount of panose canbe as high as 1-2%, which is highly undesirable since it lowers thesaccharification yield significantly.

Fermentable sugars (e.g., dextrins, monosaccharides, particularlyglucose) are produced from enzymatic saccharification. These fermentablesugars may be further purified and/or converted to useful sugarproducts. In addition, the sugars may be used as a fermentationfeedstock in a microbial fermentation process for producingend-products, such as alcohol (e.g., ethanol and butanol), organic acids(e.g., succinic acid and lactic acid), sugar alcohols (e.g., glycerol),ascorbic acid intermediates (e.g., gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g.,lysine), proteins (e.g., antibodies and fragment thereof).

In an embodiment, the fermentable sugars obtained during theliquefaction process steps are used to produce alcohol and particularlyethanol. In ethanol production, an SSF process is commonly used whereinthe saccharifying enzymes and fermenting organisms (e.g., yeast) areadded together and then carried out at a temperature of 30-40° C.

The organism used in fermentation will depend on the desiredend-product. Typically, if ethanol is the desired end product yeast willbe used as the fermenting organism. In some preferred embodiments, theethanol-producing microorganism is a yeast and specificallySaccharomyces such as strains of S. cerevisiae (U.S. Pat. No.4,316,956). A variety of S. cerevisiae are commercially available andthese include but are not limited to FALI (Fleischmann's Yeast),SUPERSTART (Alltech), FERMIOL (DSM Specialties), RED STAR (Lesaffre) andAngel alcohol yeast (Angel Yeast Company, China). The amount of starteryeast employed in the methods is an amount effective to produce acommercially significant amount of ethanol in a suitable amount of time,(e.g., to produce at least 10% ethanol from a substrate having between25-40% DS in less than 72 hours). Yeast cells are generally supplied inamounts of about 10⁴ to about 10¹², and preferably from about 10⁷ toabout 10¹⁰ viable yeast count per mL of fermentation broth. After yeastis added to the mash, it is typically subjected to fermentation forabout 24-96 hours, e.g., 35-60 hours. The temperature is between about26-34° C., typically at about 32° C., and the pH is from pH 3-6, e.g.,around pH 4-5.

The fermentation may include, in addition to a fermenting microorganisms(e.g., yeast), nutrients, and additional enzymes, including phytases.The use of yeast in fermentation is well known in the art.

In further embodiments, use of appropriate fermenting microorganisms, asis known in the art, can result in fermentation end product including,e.g., glycerol, 1,3-propanediol, gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lacticacid, amino acids, and derivatives thereof. More specifically whenlactic acid is the desired end product, a Lactobacillus sp. (L. casei)may be used; when glycerol or 1,3-propanediol are the desiredend-products E. coli may be used; and when 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid are the desired endproducts, Pantoea citrea may be used as the fermenting microorganism.The above enumerated list is only examples and one skilled in the artwill be aware of a number of fermenting microorganisms that may be usedto obtain a desired end product.

Processes for Producing Fermentation Products from UngelatinizedStarch-Containing Material

The invention relates to processes for producing fermentation productsfrom starch-containing material without gelatinization (i.e., withoutcooking) of the starch-containing material. The fermentation product,such as ethanol, can be produced without liquefying the aqueous slurrycontaining the starch-containing material and water. In one embodiment aprocess of the invention includes saccharifying (e.g., milled)starch-containing material, e.g., granular starch, below the initialgelatinization temperature, preferably in the presence of alpha-amylaseand/or carbohydrate-source generating enzyme(s) to produce sugars thatcan be fermented into the fermentation product by a suitable fermentingorganism. In this embodiment the desired fermentation product, e.g.,ethanol, is produced from ungelatinized (i.e., uncooked), preferablymilled, cereal grains, such as corn. Accordingly, in the first aspectthe invention relates to processes for producing fermentation productsfrom starch-containing material comprising simultaneously saccharifyingand fermenting starch-containing material using a carbohydrate-sourcegenerating enzyme and a fermenting organism at a temperature below theinitial gelatinization temperature of said starch-containing material.In an embodiment a protease is also present. The protease may be anyacid fungal protease or metalloprotease. The fermentation product, e.g.,ethanol, may optionally be recovered after fermentation, e.g., bydistillation. Typically amylase(s), such as glucoamylase(s) and/or othercarbohydrate-source generating enzymes, and/or alpha-amylase(s), is(are)present during fermentation. Examples of glucoamylases and othercarbohydrate-source generating enzymes include raw starch hydrolyzingglucoamylases. Examples of alpha-amylase(s) include acid alpha-amylasessuch as acid fungal alpha-amylases. Examples of fermenting organismsinclude yeast e.g., a strain of Saccharomyces cerevisiae. The term“initial gelatinization temperature” means the lowest temperature atwhich starch gelatinization commences. In general, starch heated inwater begins to gelatinize between about 50° C. and 75° C.; the exacttemperature of gelatinization depends on the specific starch and canreadily be determined by the skilled artisan. Thus, the initialgelatinization temperature may vary according to the plant species, tothe particular variety of the plant species as well as with the growthconditions. In the context of this invention the initial gelatinizationtemperature of a given starch-containing material may be determined asthe temperature at which birefringence is lost in 5% of the starchgranules using the method described by Gorinstein and Lii, 1992,Starch/Stärke 44(12): 461-466. Before initiating the process a slurry ofstarch-containing material, such as granular starch, having 10-55 w/w %dry solids (DS), preferably 25-45 w/w % dry solids, more preferably30-40 w/w % dry solids of starch-containing material may be prepared.The slurry may include water and/or process waters, such as stillage(backset), scrubber water, evaporator condensate or distillate,side-stripper water from distillation, or process water from otherfermentation product plants. Because the process of the invention iscarried out below the initial gelatinization temperature, and thus nosignificant viscosity increase takes place, high levels of stillage maybe used if desired. In an embodiment the aqueous slurry contains fromabout 1 to about 70 vol. %, preferably 15-60 vol. %, especially fromabout 30 to 50 vol. % water and/or process waters, such as stillage(backset), scrubber water, evaporator condensate or distillate,side-stripper water from distillation, or process water from otherfermentation product plants, or combinations thereof, or the like. Thestarch-containing material may be prepared by reducing the particlesize, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably0.1-0.5 mm. After being subjected to a process of the invention at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or preferably at least99% of the dry solids in the starch-containing material are convertedinto a soluble starch hydrolyzate. A process in this aspect of theinvention is conducted at a temperature below the initial gelatinizationtemperature, which means that the temperature typically lies in therange between 30-75° C., preferably between 45-60° C. In a preferredembodiment the process carried at a temperature from 25° C. to 40° C.,such as from 28° C. to 35° C., such as from 30° C. to 34° C., preferablyaround 32° C. In an embodiment the process is carried out so that thesugar level, such as glucose level, is kept at a low level, such asbelow 6 w/w %, such as below about 3 w/w %, such as below about 2 w/w %,such as below about 1 w/w %, such as below about 0.5 w/w %, or below0.25 w/w %, such as below about 0.1 w/w %. Such low levels of sugar canbe accomplished by simply employing adjusted quantities of enzyme andfermenting organism. A skilled person in the art can easily determinewhich doses/quantities of enzyme and fermenting organism to use. Theemployed quantities of enzyme and fermenting organism may also beselected to maintain low concentrations of maltose in the fermentationbroth. For instance, the maltose level may be kept below about 0.5 w/w%, such as below about 0.2 w/w %. The process of the invention may becarried out at a pH from about 3 and 7, preferably from pH 3.5 to 6, ormore preferably from pH 4 to 5. In an embodiment fermentation is ongoingfor 6 to 120 hours, in particular 24 to 96 hours.

Processes for Producing Fermentation Products from GelatinizedStarch-Containing Material

In this aspect the invention relates to processes for producingfermentation products, especially ethanol, from starch-containingmaterial, which process includes a liquefaction step and sequentially orsimultaneously performed saccharification and fermentation steps.Consequently, the invention relates to processes for producingfermentation products from starch-containing material comprising thesteps of:

(a) liquefying starch-containing material in the presence of analpha-amylase variant, or;

(b) saccharifying the liquefied material obtained in step (a) using acarbohydrate-source generating enzyme;

(c) fermenting using a fermenting organism.

In an aspect, a pullulanase such as a family GH57 pullulanase is alsoused in the liquefaction step. In an embodiment a protease, such as anacid fungal protease or a metallo protease is added before, duringand/or after liquefaction. In an embodiment the metalloprotease isderived from a strain of Thermoascus, e.g., a strain of Thermoascusaurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670. In anembodiment the carbohydrate-source generating enzyme is a glucoamylasederived from a strain of Aspergillus, e.g., Aspergillus niger orAspergillus awamori, a strain of Talaromyces, especially Talaromycesemersonii; or a strain of Athelia, especially Athelia rolfsii; a strainof Trametes, e.g., Trametes cingulata; a strain of the genusPachykytospora, e.g., a strain of Pachykytospora papyracea; or a strainof the genus Leucopaxillus, e.g., Leucopaxillus giganteus; or a strainof the genus Peniophora, e.g., a strain of the species Peniophorarufomarginata; or a mixture thereof. Saccharification step (b) andfermentation step (c) may be carried out either sequentially orsimultaneously. A pullulanase and/or metalloprotease may be added duringsaccharification and/or fermentation when the process is carried out asa sequential saccharification and fermentation process and before orduring fermentation when steps (b) and (c) are carried outsimultaneously (SSF process). The pullulanase and/or metalloprotease mayalso advantageously be added before liquefaction (pre-liquefactiontreatment), i.e., before or during step (a), and/or after liquefaction(post liquefaction treatment), i.e., after step (a). The pullulanase ismost advantageously added before or during liquefaction, i.e., before orduring step (a). The fermentation product, such as especially ethanol,may optionally be recovered after fermentation, e.g., by distillation.The fermenting organism is preferably yeast, preferably a strain ofSaccharomyces cerevisiae. In a particular embodiment, the process of theinvention further comprises, prior to step (a), the steps of:

-   -   x) reducing the particle size of the starch-containing material,        preferably by milling (e.g., using a hammer mill);    -   y) forming a slurry comprising the starch-containing material        and water.

In an embodiment the particle size is smaller than a #7 screen, e.g., a#6 screen. A #7 screen is usually used in conventional prior artprocesses. The aqueous slurry may contain from 10-55, e.g., 25-45 and30-40, w/w % dry solids (DS) of starch-containing material. The slurryis heated to above the gelatinization temperature and an alpha-amylasevariant may be added to initiate liquefaction (thinning). The slurry mayin an embodiment be jet-cooked to further gelatinize the slurry beforebeing subjected to alpha-amylase in step (a). Liquefaction may in anembodiment be carried out as a three-step hot slurry process. The slurryis heated to between 60-95° C., preferably between 70-90° C., such aspreferably between 80-85° C. at pH 4-6, preferably 4.5-5.5, andalpha-amylase variant, optionally together with a pullulanase and/orprotease, preferably metalloprotease, are added to initiate liquefaction(thinning). In an embodiment the slurry may then be jet-cooked at atemperature between 95-140° C., preferably 100-135° C., such as 105-125°C., for about 1-15 minutes, preferably for about 3-10 minutes,especially around about 5 minutes. The slurry is cooled to 60-95° C. andmore alpha-amylase variant and optionally pullulanase variant and/orprotease, preferably metalloprotease, is(are) added to finalizehydrolysis (secondary liquefaction). The liquefaction process is usuallycarried out at pH 4.0-6, in particular at a pH from 4.5 to 5.5.Saccharification step (b) may be carried out using conditions well knownin the art. For instance, a full saccharification process may last up tofrom about 24 to about 72 hours, however, it is common only to do apre-saccharification of typically 40-90 minutes at a temperature between30-65° C., typically about 60° C., followed by complete saccharificationduring fermentation in a simultaneous saccharification and fermentationprocess (SSF process). Saccharification is typically carried out attemperatures from 20-75° C., preferably from 40-70° C., typically around60° C., and at a 15 pH between 4 and 5, normally at about pH 4.5. Themost widely used process to produce a fermentation product, especiallyethanol, is a simultaneous saccharification and fermentation (SSF)process, in which there is no holding stage for the saccharification,meaning that a fermenting organism, such as yeast, and enzyme(s), may beadded together. SSF may typically be carried out at a temperature from25° C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to34° C., preferably around about 32° C. In an embodiment fermentation isongoing for 6 to 120 hours, in particular 24 to 96 hours.

Beer Making

The alpha-amylase variants may also be used in a beer-making process andsimilar fermentations; the alpha-amylases will typically be added duringthe mashing process. The process is substantially similar to themilling, liquefaction, saccharification, and fermentation processesdescribed above.

Starch Slurry Processing with Stillage

Milled starch-containing material is combined with water and recycledthin-stillage resulting in an aqueous slurry. The slurry can comprisebetween 15 to 55% ds w/w (e.g., 20 to 50%, 25 to 50%, 25 to 45%, 25 to40%, 20 to 35% and 30-36% ds). In some embodiments, the recycledthin-stillage (backset) is in the range of about 10 to 70% v/v (e.g., 10to 60%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 60%, 20 to50%, 20 to 40% and also 20 to 30%).

Once the milled starch-containing material is combined with water andbackset, the pH is not adjusted in the slurry. Further the pH is notadjusted after the addition of a phytase and optionally an alpha-amylaseto the slurry. In an embodiment, the pH of the slurry will be in therange of about pH 4.5 to less than about 6.0 (e.g., pH 4.5 to 5.8, pH4.5 to 5.6, pH 4.8 to 5.8, pH 5.0 to 5.8, pH 5.0 to 5.4, pH 5.2 to 5.5and pH 5.2 to 5.9). The pH of the slurry may be between about pH 4.5 and5.2 depending on the amount of thin stillage added to the slurry and thetype of material comprising the thin stillage. For example, the pH ofthe thin stillage may be between pH 3.8 and pH 4.5.

During ethanol production, acids can be added to lower the pH in thebeer well, to reduce the risk of microbial contamination prior todistillation.

In some embodiments, a phytase is added to the slurry. In otherembodiments, in addition to phytase, an alpha-amylase is added to theslurry. In some embodiments, a phytase and alpha-amylase are added tothe slurry sequentially. In other embodiments, a phytase andalpha-amylase are added simultaneously. In some embodiments, the slurrycomprising a phytase and optionally, an alpha-amylase, are incubated(pretreated) for a period of about 5 minutes to about 8 hours (e.g., 5minutes to 6 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, and 15minutes to 4 hours). In other embodiments, the slurry is incubated at atemperature in the range of about 40 to 115° C. (e.g., 45 to 80° C., 50to 70° C., 50 to 75° C., 60 to 110° C., 60 to 95° C., 70 to 110° C., 70to 85° C. and 77 to 86° C.).

In other embodiments, the slurry is incubated at a temperature of about0 to about 30° C. (e.g., 0 to 25° C., 0 to 20° C., 0 to 15° C., 0 to 10°C. and 0 to 5° C.) below the starch gelatinization temperature of thestarch-containing material. In some embodiments, the temperature isbelow about 68° C., below about 65° C., below about 62° C., below about60° C. and below about 55° C. In some embodiments, the temperature isabove about 45° C., above about 50° C., above about 55° C. and aboveabout 60° C. In some embodiments, the incubation of the slurrycomprising a phytase and an alpha-amylase at a temperature below thestarch gelatinization temperature is referred to as a primary (1°)liquefaction.

In one embodiment, the milled starch-containing material is corn ormilo. The slurry comprises 25 to 40% DS, the pH is in the range of 4.8to 5.2, and the slurry is incubated with a phytase and optionally analpha-amylase for 5 minutes to 2 hours, at a temperature range of 60 to75° C.

Currently, it is believed that commercially available microbialalpha-amylases used in the liquefaction process are generally not stableenough to produce liquefied starch substrate from a dry mill processusing whole ground grain at a temperature above about 80° C. at a pHlevel that is less than pH 5.6. The stability of many commerciallyavailable alpha-amylases is reduced at low pH.

In a further liquefaction step, the incubated or pretreatedstarch-containing material is exposed to an increase in temperature suchas about 0 to about 45° C. above the starch gelatinization temperatureof the starch-containing material (e.g., 70° C. to 120° C., 70° C. to110° C., and 70° C. to 90° C.) for a period of time of about 2 minutesto about 6 hours (e.g., 2 minutes to 4 hrs, 90 minutes, 140 minutes and90 to 140 minutes) at a pH of about 4.0 to 5.5 more preferably between 1hour to 2 hours. The temperature can be increased by a conventional hightemperature jet cooking system for a short period of time, for example,for 1 to 15 minutes. Then the starch may be further hydrolyzed at atemperature ranging from about 75° C. to 95° C. (e.g., 80° C. to 90° C.and 80° C. to 85° C.) for a period of about 15 to 150 minutes (e.g., 30to 120 minutes). In a preferred embodiment, the pH is not adjustedduring these process steps and the pH of the liquefied mash is in therange of about pH 4.0 to pH 5.8 (e.g., pH 4.5 to 5.8, pH 4.8 to 5.4, andpH 5.0 to 5.2). In some embodiments, a second dose of thermostablealpha-amylase is added to the secondary liquefaction step, but in otherembodiments there is no additional dosage of alpha-amylase.

The incubation and liquefaction steps may be followed bysaccharification and fermentation steps well known in the art.

Distillation

Optionally, following fermentation, an alcohol (e.g., ethanol) may beextracted by, for example, distillation and optionally followed by oneor more process steps.

In some embodiments, the yield of ethanol produced by the methodsprovided herein is at least 8%, at least 10%, at least 12%, at least14%, at least 15%, at least 16%, at least 17% and at least 18% (v/v) andat least 23% v/v. The ethanol obtained according to the process providedherein may be used as, for example, fuel ethanol, drinking ethanol,i.e., potable neutral spirits, or industrial ethanol.

By-Products

Left over from the fermentation is the grain, which is typically usedfor animal feed either in liquid or dried form. In further embodiments,the end product may include the fermentation co-products such asdistiller's dried grains (DDG) and distiller's dried grain plus solubles(DDGS), which may be used, for example, as an animal feed.

Further details on how to carry out liquefaction, saccharification,fermentation, distillation, and recovery of ethanol are well known tothe skilled person.

According to the process provided herein, the saccharification andfermentation may be carried out simultaneously or separately.

Pulp and Paper Production

The alpha-amylase variants may also be used in the production oflignocellulosic materials, such as pulp, paper and cardboard, fromstarch reinforced waste paper and cardboard, especially where re-pulpingoccurs at pH above 7 and where amylases facilitate the disintegration ofthe waste material through degradation of the reinforcing starch. Thealpha-amylase variants are especially useful in a process for producinga papermaking pulp from starch-coated printed-paper. The process may beperformed as described in WO 95/14807, comprising the following steps:

-   -   a) disintegrating the paper to produce a pulp,    -   b) treating with a starch-degrading enzyme before, during or        after step a), and    -   c) separating ink particles from the pulp after steps a) and b).

The alpha-amylase variants may also be useful in modifying starch whereenzymatically modified starch is used in papermaking together withalkaline fillers such as calcium carbonate, kaolin and clays. With thealpha-amylase variants it is possible to modify the starch in thepresence of the filler thus allowing for a simpler integrated process.

Desizing of Textiles, Fabrics and Garments

The alpha-amylase variants may also be very useful in textile, fabric orgarment desizing. In the textile processing industry, alpha-amylases aretraditionally used as auxiliaries in the desizing process to facilitatethe removal of starch-containing size, which has served as a protectivecoating on weft yarns during weaving. Complete removal of the sizecoating after weaving is important to ensure optimum results in thesubsequent processes, in which the fabric is scoured, bleached and dyed.Enzymatic starch breakdown is preferred because it does not involve anyharmful effect on the fiber material. In order to reduce processing costand increase mill throughput, the desizing process is sometimes combinedwith the scouring and bleaching steps. In such cases, non-enzymaticauxiliaries such as alkali or oxidation agents are typically used tobreak down the starch, because traditional alpha-amylases are not verycompatible with high pH levels and bleaching agents. The non-enzymaticbreakdown of the starch size leads to some fiber damage because of therather aggressive chemicals used. Accordingly, it would be desirable touse the alpha-amylase variants as they have an improved performance inalkaline solutions. The alpha-amylase variants may be used alone or incombination with a cellulase when desizing cellulose-containing fabricor textile.

Desizing and bleaching processes are well known in the art. Forinstance, such processes are described in e.g., WO 95/21247, U.S. Pat.No. 4,643,736, EP 119920, which are hereby incorporated by reference.

Cleaning Processes and Detergent Compositions

The alpha-amylase variants may be added as a component of a detergentcomposition for various cleaning or washing processes, including laundryand dishwashing. For example, the variants may be used in the detergentcompositions described in WO 96/23874 and WO 97/07202.

The alpha-amylase variants may be incorporated in detergents atconventionally employed concentrations. For example, a variant of theinvention may be incorporated in an amount corresponding to 0.00001-10mg (calculated as pure, active enzyme protein) of alpha-amylase perliter of wash/dishwash liquor using conventional dosing levels ofdetergent.

The detergent composition may for example be formulated as a hand ormachine laundry detergent composition, including a laundry additivecomposition suitable for pretreatment of stained fabrics and a rinseadded fabric softener composition or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

The detergent composition may further comprise one or more otherenzymes, such as a lipase, peroxidase, protease, another amylolyticenzyme, e.g., another alpha-amylase, glucoamylase, maltogenic amylase,CGTase, cellulase, mannanase (such as Mannaway™ from Novozymes,Denmark)), pectinase, pectin lyase, cutinase, and/or laccase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additive,e.g., a separate additive or a combined additive, can be formulated,e.g., granulate, a liquid, a slurry, etc. Preferred detergent additiveformulations are granulates, in particular non-dusting granulates,liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonyl-phenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols, fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238216.

The detergent composition may be in any convenient form, e.g., a bar, atablet, a powder, a granule, a paste or a liquid. A liquid detergent maybe aqueous, typically containing up to about 70% water and 0 to about30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of fromabout 0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide,ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0 to about 65% of a detergent builder orcomplexing agent such as zeolite, diphosphate, triphosphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates(e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly(ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleiclacrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The detergent may contain a bleaching system, which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxyben-zenesul-fonate. Alternatively, the bleaching system maycomprise peroxy acids of, e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition may be stabilized usingconventional stabilizing agents, e.g., a polyol such as propylene glycolor glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or aboric acid derivative, e.g., an aromatic borate ester, or a phenylboronic acid derivative such as 4-formylphenyl boronic acid, and thecomposition may be formulated as described in, e.g., WO 92/19708 and WO92/19709.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilre-deposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

The detergent compositions may comprise any enzyme in an amountcorresponding to 0.01-100 mg of enzyme protein per liter of wash liquor,preferably 0.055 mg of enzyme protein per liter of wash liquor, inparticular 0.1-1 mg of enzyme protein per liter of wash liquor.

One or more of the variant enzymes described herein may additionally beincorporated in the detergent formulations disclosed in WO 97/07202,which is hereby incorporated as reference.

The invention is further defined by the following embodiments:

Embodiment 1

An alpha-amylase variant comprising an alteration at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416,437 and 450 of SEQ ID NO: 1, wherein the variant has at least 60% andless than 100% sequence identity to (i) the mature polypeptide of any ofSEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii)amino acids 1 to 483 of SEQ ID NO: 1, amino acids 1 to 483 of SEQ ID NO:2, amino acids 1 to 485 of SEQ ID NO: 3, amino acids 1 to 482 of SEQ IDNO: 4, amino acids 1 to 484 of SEQ ID NO: 5, amino acids 1 to 483 of SEQID NO: 6, amino acids 1 to 485 of SEQ ID NO: 7, amino acids 1 to 485 ofSEQ ID NO: 8, amino acids 1 to 485 of SEQ ID NO: 9, amino acids 1 to 485of SEQ ID NO: 10, amino acids 1 to 485 of SEQ ID NO: 11, amino acids 1to 480 of SEQ ID NO: 12, amino acids 1 to 483 of SEQ ID NO: 13 or aminoacids 1 to 481 of SEQ ID NO: 14, and wherein the variant hasalpha-amylase activity.

Embodiment 2

The variant of Embodiment 1, which is an isolated alpha-amylase variant.

Embodiment 3

The variant of any of Embodiments 1-2, wherein the variant comprises: aninsertion or a substitution at a position corresponding to position 1with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, in particular an insertion of or asubstitution with His, Phe, Trp or Tyr, preferably His or Trp;

-   -   an insertion or a substitution at a position corresponding to        position 2 with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile,        Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, in        particular an insertion of or a substitution with His, Phe, Trp        or Tyr, preferably His or Trp;    -   a substitution at a position corresponding to position 68 with        Ala, Arg, Asn, Asp, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser,        Thr, Trp, Tyr or Val, in particular with Phr, Trp or Tyr,        preferably with Trp;    -   a substitution at a position corresponding to position 71 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with His, Phe, Trp        or Tyr, preferably with Trp;    -   a substitution at a position corresponding to position 126 with        Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with His, Phe, Trp        or Tyr, preferably with Trp;    -   a substitution at a position corresponding to position 133 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Phe, Trp or        Tyr, preferably with Tyr;    -   a substitution at a position corresponding to position 142 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Phe, Trp or        Tyr, preferably with Trp;    -   a substitution at a position corresponding to position 144 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,        Phe, Ser, Thr, Trp, Tyr or Val, in particular with His, Phe, Trp        or Tyr, preferably with Trp;    -   a substitution at a position corresponding to position 156 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,        Phe, Pro, Ser, Thr, Trp or Val, in particular with His, Phe or        Trp, preferably with Trp;    -   a substitution at a position corresponding to position 158 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,        Phe, Pro, Ser, Thr, Trp or Val, in particular with His, Phe or        Trp, preferably with Trp;    -   a substitution at a position corresponding to position 176 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Leu;    -   a substitution at a position corresponding to position 185 with        Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Pro;    -   a substitution at a position corresponding to position 201 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Tyr;    -   a substitution at a position corresponding to position 205 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Tyr;    -   a substitution at a position corresponding to position 213 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Thr;    -   a substitution at a position corresponding to position 239 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,        Phe, Pro, Thr, Trp, Tyr or Val, in particular with Ala or Gin;    -   a substitution at a position corresponding to position 279 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with His, Trp or        Tyr, preferably with Trp;    -   a substitution at a position corresponding to position 316 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Phe, Trp or        Tyr, preferably with Trp;    -   a substitution at a position corresponding to position 318 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with His, Phe, Trp        or Tyr, preferably with Trp;    -   a substitution at a position corresponding to position 360 with        Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ser;    -   a substitution at a position corresponding to position 416 with        Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Val;    -   a substitution at a position corresponding to position 437 with        Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with His, Phe, Trp        or Tyr, preferably with Trp; or    -   a substitution at a position corresponding to position 450 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Phe, Trp or        Tyr, preferably with Trp.

Embodiment 4

The variant of any of Embodiments 1-3, wherein the variant has at least65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%, butless than 100%, sequence identity to (i) the mature polypeptide of anyof SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii)amino acids 1 to 483 of SEQ ID NO: 1, amino acids 1 to 483 of SEQ ID NO:2, amino acids 1 to 485 of SEQ ID NO: 3, amino acids 1 to 482 of SEQ IDNO: 4, amino acids 1 to 484 of SEQ ID NO: 5, amino acids 1 to 483 of SEQID NO: 6, amino acids 1 to 485 of SEQ ID NO: 7, amino acids 1 to 485 ofSEQ ID NO: 8, amino acids 1 to 485 of SEQ ID NO: 9, amino acids 1 to 485of SEQ ID NO: 10, amino acids 1 to 485 of SEQ ID NO: 11, amino acids 1to 480 of SEQ ID NO: 12, amino acids 1 to 483 of SEQ ID NO: 13 or aminoacids 1 to 481 of SEQ ID NO: 14.

Embodiment 5

The variant of any of Embodiments 1-3, wherein the variant has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, but less than 100%, sequence identity to (i) the maturepolypeptide of SEQ ID NO: 1, or (ii) amino acids 1-483 of SEQ ID NO: 1.

Embodiment 6

The variant of any of Embodiments 1-3, wherein the variant has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, but less than 100%, sequence identity to (i) the maturepolypeptide of SEQ ID NO: 2, (ii) the mature polypeptide of SEQ ID NO: 2comprising the deletions I181+G182*, (iii) amino acids 1-483 of SEQ IDNO: 2, or (iv) amino acids 1-483 of SEQ ID NO: 2 comprising thedeletions I181*+G182*.

Embodiment 7

The variant of any of Embodiments 1-3, wherein the variant has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, but less than 100%, sequence identity to (i) the maturepolypeptide of SEQ ID NO: 3, (ii) the mature polypeptide of SEQ ID NO: 3comprising the deletions T183*+G184*, (iii) amino acids 1-485 of SEQ IDNO: 3, or (iv) amino acids 1-485 of SEQ ID NO: 3 comprising thedeletions T183*+G184*.

Embodiment 8

The variant of any of Embodiments 1-3, wherein the variant has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, but less than 100%, sequence identity to (i) the maturepolypeptide of SEQ ID NO: 4, (ii) the mature polypeptide of SEQ ID NO: 4comprising the deletions T180*+G181*, (iii) amino acids 1-482 of SEQ IDNO: 4, or (iv) amino acids 1-482 of SEQ ID NO: 4 comprising thedeletions T180*+G181*.

Embodiment 9

The variant of any of Embodiments 1-3, wherein the variant has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, but less than 100%, sequence identity to (i) the maturepolypeptide of SEQ ID NO: 5, (ii) the mature polypeptide of SEQ ID NO: 5comprising the deletions T182*+G183*, (iii) amino acids 1-484 of SEQ IDNO: 5, or (iv) amino acids 1-484 of SEQ ID NO: 5 comprising thedeletions T182*+G183*.

Embodiment 10

The variant of any of Embodiments 1-3, wherein the variant has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, but less than 100%, sequence identity to (i) the maturepolypeptide of SEQ ID NO: 6, (ii) the mature polypeptide of SEQ ID NO: 6comprising the deletions E178*+G179*, (iii) amino acids 1-483 of SEQ IDNO: 6, or (iv) amino acids 1-483 of SEQ ID NO: 6 comprising thedeletions E178*+G180*.

Embodiment 11

The variant of any of Embodiments 1-3, wherein the variant has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, but less than 100%, sequence identity to (i) the maturepolypeptide of SEQ ID NO: 7, (ii) the mature polypeptide of SEQ ID NO: 7comprising the deletions T183*+G184*, (iii) amino acids 1-485 of SEQ IDNO: 7, or (iv) amino acids 1-485 of SEQ ID NO: 7 comprising thedeletions T183*+G184*.

Embodiment 12

The variant of any of Embodiments 1-3, wherein the variant has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, but less than 100%, sequence identity to (i) the maturepolypeptide of SEQ ID NO: 8, (ii) the mature polypeptide of SEQ ID NO: 8comprising the deletions D183*+G184*, (iii) amino acids 1-485 of SEQ IDNO: 8, or (iv) amino acids 1-485 of SEQ ID NO: 8 comprising thedeletions D183*+G184*.

Embodiment 13

The variant of any of Embodiments 1-3, wherein the variant has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, but less than 100%, sequence identity to (i) the maturepolypeptide of SEQ ID NO: 9, (ii) the mature polypeptide of SEQ ID NO: 9comprising the deletions D183*+G184*, (iii) amino acids 1-485 of SEQ IDNO: 9, or (iv) amino acids 1-485 of SEQ ID NO: 9 comprising thedeletions D183*+G184*.

Embodiment 14

The variant of any of Embodiments 1-3, wherein the variant has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, but less than 100%, sequence identity to (i) the maturepolypeptide of SEQ ID NO: 10, (ii) the mature polypeptide of SEQ ID NO:10 comprising the deletions D183*+G184*, (iii) amino acids 1-485 of SEQID NO: 10, or (iv) amino acids 1-485 of SEQ ID NO: 10 comprising thedeletions D183*+G184*.

Embodiment 15

The variant of any of Embodiments 1-3, wherein the variant has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, but less than 100%, sequence identity to (i) the maturepolypeptide of SEQ ID NO: 11, (ii) the mature polypeptide of SEQ ID NO:11 comprising the deletions H183*+G184*, (iii) amino acids 1-485 of SEQID NO: 11, or (iv) amino acids 1-485 of SEQ ID NO: 11 comprising thedeletions H183*+G184*.

Embodiment 16

The variant of any of Embodiments 1-3, wherein the variant has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, but less than 100%, sequence identity to (i) the maturepolypeptide of SEQ ID NO: 12, or (ii) amino acids 1-480 of SEQ ID NO:12.

Embodiment 17

The variant of any of Embodiments 1-3, wherein the variant has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, but less than 100%, sequence identity to (i) the maturepolypeptide of SEQ ID NO: 13, or (ii) amino acids 1-483 of SEQ ID NO:13.

Embodiment 18

The variant of any of Embodiments 1-3, wherein the variant has at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, but less than 100%, sequence identity to (i) the maturepolypeptide of SEQ ID NO: 14, or (ii) amino acids 1-481 of SEQ ID NO:14.

Embodiment 19

The variant of any of Embodiments 1-18, which is a variant of a parentalpha-amylase selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to (i) themature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQ ID NO: 1, aminoacids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQ ID NO: 3,amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484 of SEQ ID NO:5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485 of SEQ IDNO: 7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to 485 of SEQID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids 1 to 485 ofSEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12, amino acids 1 to483 of SEQ ID NO: 13, amino acids 1 to 481 of SEQ ID NO: 14; or(b) a fragment of the mature polypeptide of any of SEQ ID NOs: 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, which has alpha-amylaseactivity.

Embodiment 20

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to (i) the maturepolypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or 13, or (ii) amino acids 1 to 483 of SEQ ID NO: 1, amino acids 1 to483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQ ID NO: 3, amino acids 1to 482 of SEQ ID NO: 4, amino acids 1 to 484 of SEQ ID NO: 5, aminoacids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485 of SEQ ID NO: 7,amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to 485 of SEQ ID NO:9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids 1 to 485 of SEQ IDNO: 11, amino acids 1 to 480 of SEQ ID NO: 12, amino acids 1 to 483 ofSEQ ID NO: 13, amino acids 1 to 481 of SEQ ID NO: 14.

Embodiment 21

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to (i) the maturepolypeptide of SEQ ID NO: 1, or (ii) amino acids 1-483 of SEQ ID NO: 1.

Embodiment 22

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to (i) the maturepolypeptide of SEQ ID NO: 2, (ii) the mature polypeptide of SEQ ID NO: 2comprising the deletions I181*+G182*, (iii) amino acids 1-483 of SEQ IDNO: 2, or (iv) amino acids 1-483 of SEQ ID NO: 2 comprising thedeletions I181*+G182*.

Embodiment 23

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to (i) the maturepolypeptide of SEQ ID NO: 3, (ii) the mature polypeptide of SEQ ID NO: 3comprising the deletions T183*+G184*, (iii) amino acids 1-485 of SEQ IDNO: 3, or (iv) amino acids 1-485 of SEQ ID NO: 3 comprising thedeletions T183*+G184*.

Embodiment 24

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to (i) the maturepolypeptide of SEQ ID NO: 4, (ii) the mature polypeptide of SEQ ID NO: 4comprising the deletions T180*+G181*, (iii) amino acids 1-482 of SEQ IDNO: 4, or (iv) amino acids 1-482 of SEQ ID NO: 4 comprising thedeletions T180*+G181*.

Embodiment 25

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 5, (ii) the mature polypeptide of SEQ ID NO: 5 comprising thedeletions T182*+G183*, (iii) amino acids 1-484 of SEQ ID NO: 5, or (iv)amino acids 1-484 of SEQ ID NO: 5 comprising the deletions T182*+G183*.

Embodiment 26

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 6, (ii) the mature polypeptide of SEQ ID NO: 6 comprising thedeletions E178*+G179*, (iii) amino acids 1-483 of SEQ ID NO: 6, or (iv)amino acids 1-483 of SEQ ID NO: 6 comprising the deletions E178*+G179*.

Embodiment 27

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 7, (ii) the mature polypeptide of SEQ ID NO: 7 comprising thedeletions T183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 7, or (iv)amino acids 1-485 of SEQ ID NO: 7 comprising the deletions T183*+G184*.

Embodiment 28

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 8, (ii) the mature polypeptide of SEQ ID NO: 8 comprising thedeletions D183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 8, or (iv)amino acids 1-485 of SEQ ID NO: 8 comprising the deletions D183*+G184*.

Embodiment 29

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 9, (ii) the mature polypeptide of SEQ ID NO: 9 comprising thedeletions D183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 9, or (iv)amino acids 1-485 of SEQ ID NO: 9 comprising the deletions D183*+G184*.

Embodiment 30

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 10, (ii) the mature polypeptide of SEQ ID NO: 10 comprising thedeletions D183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 10, or (iv)amino acids 1-485 of SEQ ID NO: 10 comprising the deletions D183*+G184*.

Embodiment 31

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 11, (ii) the mature polypeptide of SEQ ID NO: 11 comprising thedeletions H183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 11, or (iv)amino acids 1-485 of SEQ ID NO: 11 comprising the deletions H183*+G184*.

Embodiment 32

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 12, or (ii) amino acids 1-480 of SEQ ID NO: 12.

Embodiment 33

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 13, or (ii) amino acids 1-483 of SEQ ID NO: 13.

Embodiment 34

The variant of Embodiment 19, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 14, or (ii) amino acids 1-481 of SEQ ID NO: 14.

Embodiment 35

The variant of Embodiment 19, wherein the parent alpha-amylase comprisesor consists of (i) the mature polypeptide of any of SEQ ID NOs: 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) amino acids 1 to 483 ofSEQ ID NO: 1, amino acids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485of SEQ ID NO: 3, amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to484 of SEQ ID NO: 5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1to 485 of SEQ ID NO: 7, amino acids 1 to 485 of SEQ ID NO: 8, aminoacids 1 to 485 of SEQ ID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10,amino acids 1 to 485 of SEQ ID NO: 11, amino acids 1 to 480 of SEQ IDNO: 12, amino acids 1 to 483 of SEQ ID NO: 13 or amino acids 1 to 481 ofSEQ ID NO: 14.

Embodiment 36

The variant of Embodiment 19, wherein the parent alpha-amylase is afragment of the mature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13 or 14, wherein the fragment has alpha-amylaseactivity.

Embodiment 37

The variant of any of Embodiments 1 to 36, wherein the variant consistsof 300 to 700, e.g., 350 to 650, 400 to 600, 450 to 500 or 470 to 490,amino acids.

Embodiment 38

The variant of any of Embodiments 1 to 37, which comprises an alterationat two or more positions corresponding to any of positions 1, 2, 68, 71,126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316,318, 360, 416, 437 and 450 of SEQ ID NO: 1.

Embodiment 39

The variant of any of Embodiments 1 to 37, which comprises an alterationat three or more positions corresponding to any of positions 1, 2, 68,71, 126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279,316, 318, 360, 416, 437 and 450 of SEQ ID NO: 1.

Embodiment 40

The variant of any of Embodiments 1 to 37, which comprises an alterationat four or more positions corresponding to any of positions 1, 2, 68,71, 126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279,316, 318, 360, 416, 437 and 450 of SEQ ID NO: 1.

Embodiment 41

The variant of any of Embodiments 1 to 37, which comprises an alterationat five or more positions corresponding to any of positions 1, 2, 68,71, 126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279,316, 318, 360, 416, 437 and 450 of SEQ ID NO: 1.

Embodiment 42

The variant of any of Embodiments 1 to 37, which comprises an alterationat six or more positions corresponding to any of positions 1, 2, 68, 71,126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316,318, 360, 416, 437 and 450 of SEQ ID NO: 1.

Embodiment 43

The variant of any of Embodiments 1 to 37, which comprises an alterationat two or more positions corresponding to any of positions 2, 68, 133,142, 176, 185, 201, 205, 213, 360, 416 and 437 of SEQ ID NO: 1.

Embodiment 44

The variant of any of Embodiments 1 to 37, which comprises an alterationat three or more positions corresponding to any of positions 2, 68, 133,142, 176, 185, 201, 205, 213, 360, 416 and 437 of SEQ ID NO: 1.

Embodiment 45

The variant of any of Embodiments 1 to 37, which comprises an alterationat four or more positions corresponding to any of positions 2, 68, 133,142, 176, 185, 201, 205, 213, 360, 416 and 437 of SEQ ID NO: 1.

Embodiment 46

The variant of any of Embodiments 1 to 37, which comprises an alterationat five or more positions corresponding to any of positions 2, 68, 133,142, 176, 185, 201, 205, 213, 360, 416 and 437 of SEQ ID NO: 1.

Embodiment 47

The variant of any of Embodiments 1 to 37, which comprises an alterationat six or more positions corresponding to any of positions 2, 68, 133,142, 176, 185, 201, 205, 213, 360, 416 and 437 of SEQ ID NO: 1.

Embodiment 48

The variant of any of Embodiments 1 to 37, which comprises asubstitution at two or more positions corresponding to any of positions68, 176, 185, 201, 205, 213, 360, 416 and 437 of SEQ ID NO: 1.

Embodiment 49

The variant of any of Embodiments 1 to 37, which comprises asubstitution at three or more positions corresponding to any ofpositions 68, 176, 185, 201, 205, 213, 360, 416 and 437 of SEQ ID NO: 1.

Embodiment 50

The variant of any of Embodiments 1 to 37, which comprises asubstitution at four or more positions corresponding to any of positions68, 176, 185, 201, 205, 213, 360, 416 and 437 of SEQ ID NO: 1.

Embodiment 51

The variant of any of Embodiments 1 to 37, which comprises asubstitution at five or more positions corresponding to any of positions68, 176, 185, 201, 205, 213, 360, 416 and 437 of SEQ ID NO: 1.

Embodiment 52

The variant of any of Embodiments 1 to 37, which comprises asubstitution at six or more positions corresponding to any of positions68, 176, 185, 201, 205, 213, 360, 416 and 437 of SEQ ID NO: 1.

Embodiment 53

The variant of any of Embodiments 1 to 37, which comprises one or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, D416V, R437W and H450W.

Embodiment 54

The variant of any of Embodiments 1 to 37, which comprises two or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, D416V, R437W and H450W.

Embodiment 55

The variant of any of Embodiments 1 to 37, which comprises three or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, D416V, R437W and H450W.

Embodiment 56

The variant of any of Embodiments 1 to 37, which comprises four or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, D416V, R437W and H450W.

Embodiment 57

The variant of any of Embodiments 1 to 37, which comprises five or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, D416V, R437W and H450W.

Embodiment 58

The variant of any of Embodiments 1 to 37, which comprises six or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, D416V, R437W and H450W.

Embodiment 59

The variant of any of Embodiments 1 to 37, which comprises one or moresubstitutions selected from the group consisting of N2H, H68W, H133Y,H142W, K176L, E185P, I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 60

The variant of any of Embodiments 1 to 37, which comprises two or moresubstitutions selected from the group consisting of N2H, H68W, H133Y,H142W, K176L, E185P, I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 61

The variant of any of Embodiments 1 to 37, which comprises three or moresubstitutions selected from the group consisting of N2H, H68W, H133Y,H142W, K176L, E185P, I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 62

The variant of any of Embodiments 1 to 37, which comprises four or moresubstitutions selected from the group consisting of N2H, H68W, H133Y,H142W, K176L, E185P, I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 63

The variant of any of Embodiments 1 to 37, which comprises five or moresubstitutions selected from the group consisting of N2H, H68W, H133Y,H142W, K176L, E185P, I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 64

The variant of any of Embodiments 1 to 37, which comprises six or moresubstitutions selected from the group consisting of N2H, H68W, H133Y,H142W, K176L, E185P, I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 65

The variant of any of Embodiments 1 to 37, which comprises one or moresubstitutions selected from the group consisting of H68W, K176L, E185P,I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 66

The variant of any of Embodiments 1 to 37, which comprises two or moresubstitutions selected from the group consisting of H68W, K176L, E185P,I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 67

The variant of any of Embodiments 1 to 37, which comprises three or moresubstitutions selected from the group consisting of H68W, K176L, E185P,I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 68

The variant of any of Embodiments 1 to 37, which comprises four or moresubstitutions selected from the group consisting of H68W, K176L, E185P,I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 69

The variant of any of Embodiments 1 to 37, which comprises five or moresubstitutions selected from the group consisting of H68W, K176L, E185P,I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 70

The variant of any of Embodiments 1 to 37, which comprises six or moresubstitutions selected from the group consisting of H68W, K176L, E185P,I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 71

The variant of any of Embodiments 1 to 37, which comprises an insertionat a position corresponding to position 1.

Embodiment 72

The variant of any of Embodiments 1 to 37, which comprises the insertionA1AH.

Embodiment 73

The variant of any of Embodiments 1 to 37, which comprises the insertionA1AW.

Embodiment 74

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 1.

Embodiment 75

The variant of any of Embodiments 1 to 37, which comprises thesubstitution A1H.

Embodiment 76

The variant of any of Embodiments 1 to 37, which comprises thesubstitution A1W.

Embodiment 77

The variant of any of Embodiments 1 to 37, which comprises an insertionat a position corresponding to position 2.

Embodiment 78

The variant of any of Embodiments 1 to 37, which comprises the insertionN2NH.

Embodiment 79

The variant of any of Embodiments 1 to 37, which comprises the insertionN2NW

Embodiment 80

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 2.

Embodiment 81

The variant of any of Embodiments 1 to 37, which comprises thesubstitution N2H.

Embodiment 82

The variant of any of Embodiments 1 to 37, which comprises thesubstitution N2W.

Embodiment 83

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 68.

Embodiment 84

The variant of any of Embodiments 1 to 37, which comprises thesubstitution H68W.

Embodiment 85

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 71.

Embodiment 86

The variant of any of Embodiments 1 to 37, which comprises thesubstitution G71W.

Embodiment 87

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 126.

Embodiment 88

The variant of any of Embodiments 1 to 37, which comprises thesubstitution N126W.

Embodiment 89

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 133.

Embodiment 90

The variant of any of Embodiments 1 to 37, which comprises thesubstitution H133Y.

Embodiment 91

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 142.

Embodiment 92

The variant of any of Embodiments 1 to 37, which comprises thesubstitution H142W.

Embodiment 93

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 144.

Embodiment 94

The variant of any of Embodiments 1 to 37, which comprises thesubstitution P144W.

Embodiment 95

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 156.

Embodiment 96

The variant of any of Embodiments 1 to 37, which comprises thesubstitution Y156W.

Embodiment 97

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 158.

Embodiment 98

The variant of any of Embodiments 1 to 37, which comprises thesubstitution Y158W.

Embodiment 99

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 176.

Embodiment 100

The variant of any of Embodiments 1 to 37, which comprises thesubstitution K176L.

Embodiment 101

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 185.

Embodiment 102

The variant of any of Embodiments 1 to 37, which comprises thesubstitution E185P.

Embodiment 103

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 201.

Embodiment 104

The variant of any of Embodiments 1 to 37, which comprises thesubstitution I201Y.

Embodiment 105

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 205.

Embodiment 106

The variant of any of Embodiments 1 to 37, which comprises thesubstitution H205Y.

Embodiment 107

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 213.

Embodiment 108

The variant of any of Embodiments 1 to 37, which comprises thesubstitution K213T.

Embodiment 109

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 239.

Embodiment 110

The variant of any of Embodiments 1 to 37, which comprises thesubstitution S239A.

Embodiment 111

The variant of any of Embodiments 1 to 37, which comprises thesubstitution S239Q.

Embodiment 112

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 279.

Embodiment 113

The variant of any of Embodiments 1 to 37, which comprises thesubstitution F279W.

Embodiment 114

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 316.

Embodiment 115

The variant of any of Embodiments 1 to 37, which comprises thesubstitution H316W.

Embodiment 116

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 318.

Embodiment 117

The variant of any of Embodiments 1 to 37, which comprises thesubstitution L318W.

Embodiment 118

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 360.

Embodiment 119

The variant of any of Embodiments 1 to 37, which comprises thesubstitution Q360S.

Embodiment 120

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 416.

Embodiment 121

The variant of any of Embodiments 1 to 37, which comprises thesubstitution D416V.

Embodiment 122

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 437.

Embodiment 123

The variant of any of Embodiments 1 to 37, which comprises thesubstitution R437W.

Embodiment 124

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 450.

Embodiment 125

The variant of any of Embodiments 1 to 37, which comprises thesubstitution H450W.

Embodiment 126

The variant of any of Embodiment 1 to 37, which comprises a set ofsubstitutions selected from the group consisting of:

A1H+N2H,

A1H+N2W,

A1H+H68W,

A1H+G71W,

A1H+N126W,

A1H+H133Y,

A1H+H142W,

A1H+P144W,

A1H+Y156W,

A1H+Y158W,

A1H+K176L,

A1H+E185P,

A1H+I201Y,

A1H+H205Y,

A1H+K213T,

A1H+S239A,

A1H+S239Q,

A1H+F279W,

A1H+H316W,

A1H+L318W,

A1H+Q360S,

A1H+D416V,

A1H+R437W,

A1H+H450W,

A1W+N2H,

A1W+N2W,

A1W+H68W,

A1W+G71W,

A1W+N126W,

A1W+H133Y,

A1W+H142W,

A1W+P144W,

A1W+Y156W,

A1W+Y158W,

A1W+K176L,

A1W+E185P,

A1W+I201Y,

A1W+H205Y,

A1W+K213T,

A1W+S239A,

A1W+S239Q,

A1W+F279W,

A1W+H316W,

A1W+L318W,

A1W+Q360S,

A1W+D416V,

A1W+R437W,

A1W+H450W,

N2H+H68W

N2H+G71W,

N2H+N126W,

N2H+H133Y,

N2H+H142W,

N2H+P144W,

N2H+Y156W,

N2H+Y158W,

N2H+K176L,

N2H+E185P,

N2H+I201Y,

N2H+H205Y,

N2H+K213T,

N2H+S239A,

N2H+S239Q,

N2H+F279W,

N2H+H316W,

N2H+L318W,

N2H+Q360S,

N2H+D416V,

N2H+R437W,

N2H+H450W,

N2W+H68W

N2W+G71W,

N2W+N126W,

N2W+H133Y,

N2W+H142W,

N2W+P144W,

N2W+Y156W,

N2W+Y158W,

N2W+K176L,

N2W+E185P,

N2W+I201Y,

N2W+H205Y,

N2W+K213T,

N2W+S239A,

N2W+S239Q,

N2W+F279W,

N2W+H316W,

N2W+L318W,

N2W+Q360S,

N2W+D416V,

N2W+R437W,

N2W+H450W,

H68W+G71W,

H68W+N126W,

H68W+H133Y,

H68W+H142W,

H68W+P144W,

H68W+Y156W,

H68W+Y158W,

H68W+K176L,

H68W+E185P,

H68W+I201Y,

H68W+H205Y,

H68W+K213T,

H68W+S239A,

H68W+S239Q,

H68W+F279W,

H68W+H316W,

H68W+L318W,

H68W+Q360S,

H68W+D416V,

H68W+R437W,

H68W+H450W,

G71W+N126W,

G71W+H133Y,

G71W+H142W,

G71W+P144W,

G71W+Y156W,

G71W+Y158W,

G71W+K176L,

G71W+E185P,

G71W+I201Y,

G71W+H205Y,

G71W+K213T,

G71W+S239A,

G71W+S239Q,

G71W+F279W,

G71W+H316W,

G71W+L318W,

G71W+Q360S,

G71W+D416V,

G71W+R437W,

G71W+H450W,

N126W+H133Y,

N126W+H142W,

N126W+P144W,

N126W+Y156W,

N126W+Y158W,

N126W+K176L,

N126W+E185P,

N126W+I201Y,

N126W+H205Y,

N126W+K213T,

N126W+S239A,

N126W+S239Q,

N126W+F279W,

N126W+H316W,

N126W+L318W,

N126W+Q360S,

N126W+D416V,

N126W+R437W,

N126W+H450W,

H133Y+H142W,

H133Y+P144W,

H133Y+Y156W,

H133Y+Y158W,

H133Y+K176L,

H133Y+E185P,

H133Y+I201Y,

H133Y+H205Y,

H133Y+K213T,

H133Y+S239A,

H133Y+S239Q,

H133Y+F279W,

H133Y+H316W,

H133Y+L318W,

H133Y+Q360S,

H133Y+D416V,

H133Y+R437W,

H133Y+H450W,

H142W+P144W,

H142W+Y156W,

H142W+Y158W,

H142W+K176L,

H142W+E185P,

H142W+I201Y,

H142W+H205Y,

H142W+K213T,

H142W+S239A,

H142W+S239Q,

H142W+F279W,

H142W+H316W,

H142W+L318W,

H142W+Q360S,

H142W+D416V,

H142W+R437W,

H142W+H450W,

P144W+Y156W,

P144W+Y158W,

P144W+K176L,

P144W+E185P,

P144W+I201Y,

P144W+H205Y,

P144W+K213T,

P144W+S239A,

P144W+S239Q,

P144W+F279W,

P144W+H316W,

P144W+L318W,

P144W+Q360S,

P144W+D416V,

P144W+R437W,

P144W+H450W,

Y156W+Y158W,

Y156W+K176L,

Y156W+E185P,

Y156W+I201Y,

Y156W+H205Y,

Y156W+K213T,

Y156W+S239A,

Y156W+S239Q,

Y156W+F279W,

Y156W+H316W,

Y156W+L318W,

Y156W+Q360S,

Y156W+D416V,

Y156W+R437W,

Y156W+H450W,

Y158W+K176L,

Y158W+E185P,

Y158W+I201Y,

Y158W+H205Y,

Y158W+K213T,

Y158W+S239A,

Y158W+S239Q,

Y158W+F279W,

Y158W+H316W,

Y158W+L318W,

Y158W+Q360S,

Y158W+D416V,

Y158W+R437W,

Y158W+H450W,

K176L+E185P,

K176L+I201Y,

K176L+H205Y,

K176L+K213T,

K176L+S239A,

K176L+S239Q,

K176L+F279W,

K176L+H316W,

K176L+L318W,

K176L+Q360S,

K176L+D416V,

K176L+R437W,

K176L+H450W,

E185P+I201Y,

E185P+H205Y,

E185P+K213T,

E185P+S239A,

E185P+S239,

E185P+F279W,

E185P+H316W

E185P+L318W,

E185P+Q360S,

E185P+D416V,

E185P+R437W,

E185P+H450W,

I201Y+H205Y,

I201Y+K213T,

I201Y+S239A,

I201Y+S239Q,

I201Y+F279W,

I201Y+H316W,

I201Y+L318W,

I201Y+Q360S,

I201Y+D416V,

I201Y+R437W,

I201Y+H450W,

H205Y+K213T,

H205Y+S239A,

H205Y+S239Q,

H205Y+F279W,

H205Y+H316W,

H205Y+L318W,

H205Y+Q360S,

H205Y+D416V,

H205Y+R437W,

H205Y+H450W,

K213T+S239A,

K213T+S239Q,

K213T+F279W,

K213T+H316W,

K213T+L318W,

K213T+Q360S,

K213T+D416V,

K213T+R437W,

K213T+H450W,

S239A+F279W,

S239A+H316W,

S239A+L318W,

S239A+Q360S,

S239A+D416V,

S239A+R437W,

S239A+H450W,

S239Q+F279W,

S239Q+H316W,

S239Q+L318W,

S239Q+Q360S,

S239Q+D416V,

S239Q+R437W,

S239Q+H450W,

F279W+H316W,

F279W+L318W,

F279W+Q360S,

F279W+D416V,

F279W+R437W,

F279W+H450W,

H316W+L318W,

H316W+Q360S,

H316W+D416V,

H316W+R437W,

H316W+H450W,

L318W+Q360S,

L318W+D416V,

L318W+R437W,

L318W+H450W,

Q360S+D416V,

Q360S+R437W,

Q360S+H450W,

D416V+R437W,

D416V+H450W, and

R437W+H450W.

Embodiment 127

The variant of any of Embodiments 1 to 37, which comprises an alterationat a position corresponding to position 1, in particular the insertionA1AH or A1AW or the substitution A1H or A1W, in combination with analteration at one or more positions corresponding to any of positions 2,68, 71, 126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279,316, 318, 360, 416, 437 and 450, in particular one or more alterationsselected from the group consisting of N2NH, N2NW, N2H, N2W, H68W, G71W,N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W.

Embodiment 128

The variant of any of Embodiments 1 to 37, which comprises an alterationat a position corresponding to position 1, in particular the insertionA1AH or A1AW or the substitution A1H or A1W, in combination with analteration at two or more positions corresponding to any of positions 2,68, 71, 126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279,316, 318, 360, 416, 437 and 450, in particular two or more alterationsselected from the group consisting of N2NH, N2NW, N2H, N2W, H68W, G71W,N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W.

Embodiment 129

The variant of any of Embodiments 1 to 37, which comprises an alterationat a position corresponding to position 2, in particular the insertionN2NH or N2NW or the substitution N2H or N2W, preferably the substitutionN2H, in combination with an alteration at one or more positionscorresponding to any of positions 1, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450,in particular one or more alterations selected from the group consistingof A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, H68F, H68Y, H68W, G71F,G71H, G71Y, G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W,H142F, H142Y, H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W,Y158F, Y158H, Y158W, K176L, E185P, I201F, I201Y, H205Y, K213T, S239A,S239Q, F279Y, F279W, H316F, H316Y, H316W, L318F, L318H, L318Y, L318W,Q360S, D416V, R437F, R437H, R437Y, R437W, H450F, H450Y and H450W,preferably one or more alterations selected from the group consisting ofA1AH, A1AW, A1H, A1W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, D416V, R437W and H450W.

Embodiment 130

The variant of any of Embodiments 1 to 37, which comprises an alterationat a position corresponding to position 2, in particular the insertionN2NH or N2NW or the substitution N2H or N2W, preferably the substitutionN2H, in combination with a substitution at one or more positionscorresponding to any of positions 68, 176, 185, 201, 205, 213, 360, 416and 437, in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, H205Y, K213T, Q360S, D416V andR437W.

Embodiment 131

The variant of any of Embodiments 1 to 37, which comprises an alterationat a position corresponding to position 2, in particular the insertionN2NH or N2NW or the substitution N2H or N2W, preferably the substitutionN2H, in combination with a substitution at one or more positionscorresponding to any of positions 68, 133, 142, 156, 158, 279 and 437,in particular one or more substitutions selected from the groupconsisting of H68W, H133Y, H142W, Y156W, Y158W, F279W and R437W.

Embodiment 132

The variant of any of Embodiments 1 to 37, which comprises an alterationat a position corresponding to position 2, in particular the insertionN2NH or N2NW or the substitution N2H or N2W, preferably the substitutionN2H, in combination with an alteration at two or more positionscorresponding to any of positions 1, 68, 71, 126, 133, 142, 144, 156,158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450,in particular two or more alterations selected from the group consistingof A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, H68F, H68Y, H68W, G71F,G71H, G71Y, G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W,H142F, H142Y, H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W,Y158F, Y158H, Y158W, K176L, E185P, I201F, I201Y, H205Y, K213T, S239A,S239Q, F279Y, F279W, H316F, H316Y, H316W, L318F, L318H, L318Y, L318W,Q360S, D416V, R437F, R437H, R437Y, R437W, H450F, H450Y and H450W,preferably two or more alterations selected from the group consisting ofA1AH, A1AW, A1H, A1W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, D416V, R437W and H450W.

Embodiment 133

The variant of any of Embodiments 1 to 37, which comprises an alterationat a position corresponding to position 2, in particular the insertionN2NH or N2NW or the substitution N2H or N2W, preferably the substitutionN2H, in combination with a substitution at two or more positionscorresponding to any of positions 68, 176, 185, 201, 205, 213, 360, 416and 437, in particular two or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, H205Y, K213T, Q360S, D416V andR437W.

Embodiment 134

The variant of any of Embodiments 1 to 37, which comprises an alterationat a position corresponding to position 2, in particular the insertionN2NH or N2NW or the substitution N2H or N2W, preferably the substitutionN2H, in combination with a substitution at two or more positionscorresponding to any of positions 68, 133, 142, 156, 158, 279 and 437,in particular two or more substitutions selected from the groupconsisting of H68W, H133Y, H142W, Y156W, Y158W, F279W and R437W.

Embodiment 135

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 68, in particularthe substitution H68W, in combination with an alteration at one or morepositions corresponding to any of positions 1, 2, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416,437 and 450, in particular one or more alterations selected from thegroup consisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH,N2N2F, N2NY, N2NW, N2H, N2F, N2Y, N2W, G71F, G71H, G71Y, G71W, N126F,N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y, H142W, P144F,P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H, Y158W, K176L,E185P, I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F,H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H,R437Y, R437W, H450F, H450Y and H450W, preferably one or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW,N2H, N2W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V,R437W and H450W.

Embodiment 136

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 68, in particularthe substitution H68W, in combination with a substitution at one or morepositions corresponding to any of positions 176, 185, 201, 205, 213,360, 416 and 437, in particular one or more substitutions selected fromthe group consisting of K176L, E185P, I201Y, H205Y, K213T, Q360S, D416Vand R437W.

Embodiment 137

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 68, in particularthe substitution H68W, in combination with a substitution at one or morepositions corresponding to any of positions 2, 133, 142, 156, 158, 279and 437, in particular one or more substitutions selected from the groupconsisting of N2H, H133Y, H142W, Y156W, Y158W, F279W and R437W.

Embodiment 138

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 68, in particularthe substitution H68W, in combination with an alteration at two or morepositions corresponding to any of positions 1, 2, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416,437 and 450, in particular two or more alterations selected from thegroup consisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH,N2N2F, N2NY, N2NW, N2H, N2F, N2Y, N2W, G71F, G71H, G71Y, G71W, N126F,N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y, H142W, P144F,P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H, Y158W, K176L,E185P, I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F,H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H,R437Y, R437W, H450F, H450Y and H450W, preferably two or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW,N2H, N2W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V,R437W and H450W.

Embodiment 139

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 68, in particularthe substitution H68W, in combination with a substitution at two or morepositions corresponding to any of positions 176, 185, 201, 205, 213,360, 416 and 437, in particular two or more substitutions selected fromthe group consisting of K176L, E185P, I201Y, H205Y, K213T, Q360S, D416Vand R437W.

Embodiment 140

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 68, in particularthe substitution H68W, in combination with a substitution at two or morepositions corresponding to any of positions 2, 133, 142, 156, 158, 279and 437, in particular two or more substitutions selected from the groupconsisting of N2H, H133Y, H142W, Y156W, Y158W, F279W and R437W.

Embodiment 141

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 71, in particularthe substitution G71W, in combination with a substitution at one or morepositions corresponding to any of positions 1, 2, 68, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416,437 and 450, in particular one or more substitutions selected from thegroup consisting of A1H, A1W, N2H, N2W, H68W, N126W, H133Y, H142W,P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q,F279W, H316W, L318W, Q360S, D416V, R437W and H450W.

Embodiment 142

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 71, in particularthe substitution G71W, in combination with a substitution at two or morepositions corresponding to any of positions 1, 2, 68, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416,437 and 450, in particular two or more substitutions selected from thegroup consisting of A1H, A1W, N2H, N2W, H68W, N126W, H133Y, H142W,P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q,F279W, H316W, L318W, Q360S, D416V, R437W and H450W.

Embodiment 143

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 126, in particularthe substitution N126W, in combination with a substitution at one ormore positions corresponding to any of positions 1, 2, 68, 71, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416,437 and 450, in particular one or more substitutions selected from thegroup consisting of A1H, A1W, N2H, N2W, H68W, G71W, H133Y, H142W, P144W,Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W,H316W, L318W, Q360S, D416V, R437W and H450W.

Embodiment 144

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 126, in particularthe substitution N126W, in combination with a substitution at two ormore positions corresponding to any of positions 1, 2, 68, 71, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416,437 and 450, in particular two or more substitutions selected from thegroup consisting of A1H, A1W, N2H, N2W, H68W, G71W, H133Y, H142W, P144W,Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W,H316W, L318W, Q360S, D416V, R437W and H450W.

Embodiment 145

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 133, in particularthe substitution H133Y, in combination with an alteration at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 142, 144,156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular one or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H142F, H142Y, H142W, P144F, P144H,P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H, Y158W, K176L, E185P,I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F, H316Y,H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H, R437Y,R437W, H450F, H450Y and H450W, preferably one or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW,N2H, N2W, H68W, G71W, N126W, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V,R437W and H450W.

Embodiment 146

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 133, in particularthe substitution H133Y, in combination with a substitution at one ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 360, 416 and 437, in particular one or more substitutions selectedfrom the group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T,Q360S, D416V and R437W.

Embodiment 147

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 133, in particularthe substitution H133Y, in combination with a substitution at one ormore positions corresponding to any of positions 2, 68, 142, 156, 158,279 and 437, in particular one or more substitutions selected from thegroup consisting of N2H, H68W, H142W, Y156W, Y158W, F279W and R437W.

Embodiment 148

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 133, in particularthe substitution H133Y, in combination with an alteration at two or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 142, 144,156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular two or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H142F, H142Y, H142W, P144F, P144H,P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H, Y158W, K176L, E185P,I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F, H316Y,H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H, R437Y,R437W, H450F, H450Y and H450W, preferably two or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW,N2H, N2W, H68W, G71W, N126W, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V,R437W and H450W.

Embodiment 149

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 133, in particularthe substitution H133Y, in combination with a substitution at two ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 360, 416 and 437, in particular two or more substitutions selectedfrom the group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T,Q360S, D416V and R437W.

Embodiment 150

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 133, in particularthe substitution H133Y, in combination with a substitution at two ormore positions corresponding to any of positions 2, 68, 142, 156, 158,279 and 437, in particular two or more substitutions selected from thegroup consisting of N2H, H68W, H142W, Y156W, Y158W, F279W and R437W.

Embodiment 151

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 142, in particularthe substitution H142W, in combination with an alteration at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 144,156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular one or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, P144F, P144H,P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H, Y158W, K176L, E185P,I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F, H316Y,H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H, R437Y,R437W, H450F, H450Y and H450W, preferably one or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW,N2H, N2W, H68W, G71W, N126W, H133Y, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V,R437W and H450W.

Embodiment 152

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 142, in particularthe substitution H142W, in combination with a substitution at one ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 360, 416 and 437, in particular one or more substitutions selectedfrom the group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T,Q360S, D416V and R437W.

Embodiment 153

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 142, in particularthe substitution H142W, in combination with a substitution at one ormore positions corresponding to any of positions 2, 68, 133, 156, 158,279 and 437, in particular one or more substitutions selected from thegroup consisting of N2H, H68W, H133Y, Y156W, Y158W, F279W and R437W.

Embodiment 154

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 142, in particularthe substitution H142W, in combination with an alteration at two or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 144,156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular two or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, P144F, P144H,P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H, Y158W, K176L, E185P,I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F, H316Y,H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H, R437Y,R437W, H450F, H450Y and H450W, preferably two or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW,N2H, N2W, H68W, G71W, N126W, H133Y, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V,R437W and H450W.

Embodiment 155

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 142, in particularthe substitution H142W, in combination with a substitution at two ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 360, 416 and 437, in particular two or more substitutions selectedfrom the group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T,Q360S, D416V and R437W.

Embodiment 156

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 142, in particularthe substitution H142W, in combination with a substitution at two ormore positions corresponding to any of positions 2, 68, 133, 156, 158,279 and 437, in particular two or more substitutions selected from thegroup consisting of N2H, H68W, H133Y, Y156W, Y158W, F279W and R437W.

Embodiment 157

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 144, in particularthe substitution P144W, in combination with a substitution at one ormore positions corresponding to any of positions 1, 2, 68, 71, 126, 133,142, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416,437 and 450, in particular one or more substitutions selected from thegroup consisting of A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W,Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W,H316W, L318W, Q360S, D416V, R437W and H450W.

Embodiment 158

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 144, in particularthe substitution P144W, in combination with a substitution at two ormore positions corresponding to any of positions 1, 2, 68, 71, 126, 133,142, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416,437 and 450, in particular two or more substitutions selected from thegroup consisting of A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W,Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W,H316W, L318W, Q360S, D416V, R437W and H450W.

Embodiment 159 Embodiment 160

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 156, in particularthe substitution Y156W, in combination with an alteration at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular one or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y158F, Y158H, Y158W, K176L, E185P,I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F, H316Y,H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H, R437Y,R437W, H450F, H450Y and H450W, preferably one or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW,N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V,R437W and H450W.

Embodiment 161

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 156, in particularthe substitution Y156W, in combination with a substitution at one ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 360, 416 and 437, in particular one or more substitutions selectedfrom the group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T,Q360S, D416V and R437W.

Embodiment 162

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 156, in particularthe substitution Y156W, in combination with a substitution at one ormore positions corresponding to any of positions 2, 68, 133, 142, 158,279 and 437, in particular one or more substitutions selected from thegroup consisting of N2H, H68W, H133Y, H142W, Y158W, F279W and R437W.

Embodiment 163

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 156, in particularthe substitution Y156W, in combination with an alteration at two or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular two or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y158F, Y158H, Y158W, K176L, E185P,I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F, H316Y,H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H, R437Y,R437W, H450F, H450Y and H450W, preferably two or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW,N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V,R437W and H450W.

Embodiment 164

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 156, in particularthe substitution Y156W, in combination with a substitution at two ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 360, 416 and 437, in particular two or more substitutions selectedfrom the group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T,Q360S, D416V and R437W.

Embodiment 165

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 156, in particularthe substitution Y156W, in combination with a substitution at two ormore positions corresponding to any of positions 2, 68, 133, 142, 158,279 and 437, in particular two or more substitutions selected from thegroup consisting of N2H, H68W, H133Y, H142W, Y158W, F279W and R437W.

Embodiment 166

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 158, in particularthe substitution Y158W, in combination with an alteration at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular one or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, K176L, E185P,I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F, H316Y,H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H, R437Y,R437W, H450F, H450Y and H450W, preferably one or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW,N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V,R437W and H450W.

Embodiment 167

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 158, in particularthe substitution Y158W, in combination with a substitution at one ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 360, 416 and 437, in particular one or more substitutions selectedfrom the group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T,Q360S, D416V and R437W.

Embodiment 168

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 158, in particularthe substitution Y158W, in combination with a substitution at one ormore positions corresponding to any of positions 2, 68, 133, 142, 156,279 and 437, in particular one or more substitutions selected from thegroup consisting of N2H, H68W, H133Y, H142W, Y156W, F279W and R437W.

Embodiment 169

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 158, in particularthe substitution Y158W, in combination with an alteration at two or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular two or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, K176L, E185P,I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F, H316Y,H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H, R437Y,R437W, H450F, H450Y and H450W, preferably two or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW,N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V,R437W and H450W.

Embodiment 170

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 158, in particularthe substitution Y158W, in combination with a substitution at two ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 360, 416 and 437, in particular two or more substitutions selectedfrom the group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T,Q360S, D416V and R437W.

Embodiment 171

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 158, in particularthe substitution Y158W, in combination with a substitution at two ormore positions corresponding to any of positions 2, 68, 133, 142, 156,279 and 437, in particular two or more substitutions selected from thegroup consisting of N2H, H68W, H133Y, H142W, Y156W, F279W and R437W.

Embodiment 172

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 176, in particularthe substitution K176L, in combination with an alteration at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular one or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, E185P, I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W,H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F,R437H, R437Y, R437W, H450F, H450Y and H450W, preferably one or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W.

Embodiment 173

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 176, in particularthe substitution K176L, in combination with a substitution at one ormore positions corresponding to any of positions 68, 185, 201, 205, 213,360, 416 and 437, in particular one or more substitutions selected fromthe group consisting of H68W, E185P, I201Y, H205Y, K213T, Q360S, D416Vand R437W.

Embodiment 174

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 176, in particularthe substitution K176L, in combination with a substitution at one ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 279 and 437, in particular one or more substitutions selected fromthe group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W andR437W.

Embodiment 175

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 176, in particularthe substitution K176L, in combination with an alteration at two or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular two or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, E185P, I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W,H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F,R437H, R437Y, R437W, H450F, H450Y and H450W, preferably two or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W.

Embodiment 176

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 176, in particularthe substitution K176L, in combination with a substitution at two ormore positions corresponding to any of positions 68, 185, 201, 205, 213,360, 416 and 437, in particular two or more substitutions selected fromthe group consisting of H68W, E185P, I201Y, H205Y, K213T, Q360S, D416Vand R437W.

Embodiment 177

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 176, in particularthe substitution K176L, in combination with a substitution at two ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 279 and 437, in particular two or more substitutions selected fromthe group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W andR437W.

Embodiment 178

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 185, in particularthe substitution E185P, in combination with an alteration at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular one or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W,H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F,R437H, R437Y, R437W, H450F, H450Y and H450W, preferably one or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W.

Embodiment 179

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 185, in particularthe substitution E185P, in combination with a substitution at one ormore positions corresponding to any of positions 68, 176, 201, 205, 213,360, 416 and 437, in particular one or more substitutions selected fromthe group consisting of H68W, K176L, I201Y, H205Y, K213T, Q360S, D416Vand R437W.

Embodiment 180

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 185, in particularthe substitution E185P, in combination with a substitution at one ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 279 and 437, in particular one or more substitutions selected fromthe group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W andR437W.

Embodiment 181

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 185, in particularthe substitution E185P, in combination with an alteration at two or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 201, 205, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular two or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y, F279W,H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F,R437H, R437Y, R437W, H450F, H450Y and H450W, preferably two or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W.

Embodiment 182

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 185, in particularthe substitution E185P, in combination with a substitution at two ormore positions corresponding to any of positions 68, 176, 201, 205, 213,360, 416 and 437, in particular two or more substitutions selected fromthe group consisting of H68W, K176L, I201Y, H205Y, K213T, Q360S, D416Vand R437W.

Embodiment 183

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 185, in particularthe substitution E185P, in combination with a substitution at two ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 279 and 437, in particular two or more substitutions selected fromthe group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W andR437W.

Embodiment 184

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 201, in particularthe substitution I201Y, in combination with an alteration at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 205, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular one or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, E185P, H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F,H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H,R437Y, R437W, H450F, H450Y and H450W, preferably one or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW,N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L,E185P, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V,R437W and H450W.

Embodiment 185

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 201, in particularthe substitution I201Y, in combination with a substitution at one ormore positions corresponding to any of positions 68, 176, 185, 205, 213,360, 416 and 437, in particular one or more substitutions selected fromthe group consisting of H68W, K176L, E185P, H205Y, K213T, Q360S, D416Vand R437W.

Embodiment 186

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 201, in particularthe substitution I201Y, in combination with a substitution at one ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 279 and 437, in particular one or more substitutions selected fromthe group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W andR437W.

Embodiment 187

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 201, in particularthe substitution I201Y, in combination with an alteration at two or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 205, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular two or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, E185P, H205Y, K213T, S239A, S239Q, F279Y, F279W, H316F,H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H,R437Y, R437W, H450F, H450Y and H450W, preferably two or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW,N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L,E185P, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V,R437W and H450W.

Embodiment 188

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 201, in particularthe substitution I201Y, in combination with a substitution at two ormore positions corresponding to any of positions 68, 176, 185, 205, 213,360, 416 and 437, in particular two or more substitutions selected fromthe group consisting of H68W, K176L, E185P, H205Y, K213T, Q360S, D416Vand R437W.

Embodiment 189

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 201, in particularthe substitution I201Y, in combination with a substitution at two ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 279 and 437, in particular two or more substitutions selected fromthe group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W andR437W.

Embodiment 190

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 205, in particularthe substitution H205Y, in combination with an alteration at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular one or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, E185P, I201F, I201Y, K213T, S239A, S239Q, F279Y, F279W,H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F,R437H, R437Y, R437W, H450F, H450Y and H450W, preferably one or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W.

Embodiment 191

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 205, in particularthe substitution H205Y, in combination with a substitution at one ormore positions corresponding to any of positions 68, 176, 185, 201, 213,360, 416 and 437, in particular one or more substitutions selected fromthe group consisting of H68W, K176L, E185P, I201Y, K213T, Q360S, D416Vand R437W.

Embodiment 192

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 205, in particularthe substitution H205Y, in combination with a substitution at one ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 279 and 437, in particular one or more substitutions selected fromthe group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W andR437W.

Embodiment 193

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 205, in particularthe substitution H205Y, in combination with an alteration at two or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 213, 239, 279, 316, 318, 360, 416, 437 and450, in particular two or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, E185P, I201F, I201Y, K213T, S239A, S239Q, F279Y, F279W,H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F,R437H, R437Y, R437W, H450F, H450Y and H450W, preferably two or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, K213T, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W.

Embodiment 194

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 205, in particularthe substitution H205Y, in combination with a substitution at two ormore positions corresponding to any of positions 68, 176, 185, 201, 213,360, 416 and 437, in particular two or more substitutions selected fromthe group consisting of H68W, K176L, E185P, I201Y, K213T, Q360S, D416Vand R437W.

Embodiment 195

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 205, in particularthe substitution H205Y, in combination with a substitution at two ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 279 and 437, in particular two or more substitutions selected fromthe group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W andR437W.

Embodiment 196

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 213, in particularthe substitution K213T, in combination with an alteration at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 239, 279, 316, 318, 360, 416, 437 and450, in particular one or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, E185P, I201F, I201Y, H205Y, S239A, S239Q, F279Y, F279W,H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F,R437H, R437Y, R437W, H450F, H450Y and H450W, preferably one or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W.

Embodiment 197

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 213, in particularthe substitution K213T, in combination with a substitution at one ormore positions corresponding to any of positions 68, 176, 185, 201, 205,360, 416 and 437, in particular one or more substitutions selected fromthe group consisting of H68W, K176L, E185P, I201Y, H205Y, Q360S, D416Vand R437W.

Embodiment 198

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 213, in particularthe substitution K213T, in combination with a substitution at one ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 279 and 437, in particular one or more substitutions selected fromthe group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W andR437W.

Embodiment 199

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 213, in particularthe substitution K213T, in combination with an alteration at two or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 239, 279, 316, 318, 360, 416, 437 and450, in particular two or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, E185P, I201F, I201Y, H205Y, S239A, S239Q, F279Y, F279W,H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F,R437H, R437Y, R437W, H450F, H450Y and H450W, preferably two or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, S239A, S239Q, F279W, H316W, L318W,Q360S, D416V, R437W and H450W.

Embodiment 200

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 213, in particularthe substitution K213T, in combination with a substitution at two ormore positions corresponding to any of positions 68, 176, 185, 201, 205,360, 416 and 437, in particular two or more substitutions selected fromthe group consisting of H68W, K176L, E185P, I201Y, H205Y, Q360S, D416Vand R437W.

Embodiment 201

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 213, in particularthe substitution K213T, in combination with a substitution at two ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 279 and 437, in particular two or more substitutions selected fromthe group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W andR437W.

Embodiment 202

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 239, in particularthe substitution S239A or S239Q, in combination with a substitution atone or more positions corresponding to any of positions 1, 2, 68, 71,126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 279, 316, 318,360, 416, 437 and 450, in particular one or more substitutions selectedfrom the group consisting of A1H, A1W, N2H, N2W, H68W, G71W, N126W,H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T,F279W, H316W, L318W, Q360S, D416V, R437W and H450W.

Embodiment 203

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 239, in particularthe substitution S239A or S239Q, in combination with a substitution attwo or more positions corresponding to any of positions 1, 2, 68, 71,126, 133, 142, 144, 156, 158, 176, 185, 201, 205, 213, 279, 316, 318,360, 416, 437 and 450, in particular two or more substitutions selectedfrom the group consisting of A1H, A1W, N2H, N2W, H68W, G71W, N126W,H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T,F279W, H316W, L318W, Q360S, D416V, R437W and H450W.

Embodiment 204

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 279, in particularthe substitution F279W, in combination with an alteration at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 316, 318, 360, 416, 437 and450, in particular one or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, E185P, I201F, I201Y, H205Y, K213T, S239A, S239Q, H316F,H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H,R437Y, R437W, H450F, H450Y and H450W, preferably one or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW,N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L,E185P, I201Y, H205Y, K213T, S239A, S239Q, H316W, L318W, Q360S, D416V,R437W and H450W.

Embodiment 205

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 279, in particularthe substitution F279W, in combination with a substitution at one ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 360, 416 and 437, in particular one or more substitutions selectedfrom the group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T,Q360S, D416V and R437W.

Embodiment 206

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 279, in particularthe substitution F279W, in combination with a substitution at one ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158 and 437, in particular one or more substitutions selected from thegroup consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, and R437W.

Embodiment 207

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 279, in particularthe substitution F279W, in combination with an alteration at two or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 316, 318, 360, 416, 437 and450, in particular two or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, E185P, I201F, I201Y, H205Y, K213T, S239A, S239Q, H316F,H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V, R437F, R437H,R437Y, R437W, H450F, H450Y and H450W, preferably two or more alterationsselected from the group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW,N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L,E185P, I201Y, H205Y, K213T, S239A, S239Q, H316W, L318W, Q360S, D416V,R437W and H450W.

Embodiment 208

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 279, in particularthe substitution F279W, in combination with a substitution at two ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 360, 416 and 437, in particular two or more substitutions selectedfrom the group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T,Q360S, D416V and R437W.

Embodiment 209

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 279, in particularthe substitution F279W, in combination with a substitution at two ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158 and 437, in particular two or more substitutions selected from thegroup consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W and R437W.

Embodiment 210

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 316, in particularthe substitution H316W, in combination with a substitution at one ormore positions corresponding to any of positions 1, 2, 68, 71, 126, 133,142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 318, 360, 416,437 and 450, in particular one or more substitutions selected from thegroup consisting of A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W,P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q,F279W, L318W, Q360S, D416V, R437W and H450W.

Embodiment 211

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 316, in particularthe substitution H316W, in combination with a substitution at two ormore positions corresponding to any of positions 1, 2, 68, 71, 126, 133,142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 318, 360, 416,437 and 450, in particular two or more substitutions selected from thegroup consisting of A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W,P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q,F279W, L318W, Q360S, D416V, R437W and H450W.

Embodiment 212

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 318, in particularthe substitution L318W, in combination with a substitution at one ormore positions corresponding to any of positions 1, 2, 68, 71, 126, 133,142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 360, 416,437 and 450, in particular one or more substitutions selected from thegroup consisting of A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W,P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q,F279W, H316W, Q360S, D416V, R437W and H450W.

Embodiment 213

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 318, in particularthe substitution L318W, in combination with a substitution at two ormore positions corresponding to any of positions 1, 2, 68, 71, 126, 133,142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 360, 416,437 and 450, in particular two or more substitutions selected from thegroup consisting of A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W,P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q,F279W, H316W, Q360S, D416V, R437W and H450W.

Embodiment 214

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 360, in particularthe substitution Q360S, in combination with an alteration at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 416, 437 and450, in particular one or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, E185P, I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y,F279W, H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, D416V, R437F,R437H, R437Y, R437W, H450F, H450Y and H450W, preferably one or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, D416V, R437W and H450W.

Embodiment 215

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 360, in particularthe substitution Q360S, in combination with a substitution at one ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 416 and 437, in particular one or more substitutions selected fromthe group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T, D416Vand R437W.

Embodiment 216

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 360, in particularthe substitution Q360S, in combination with a substitution at one ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 279 and 437, in particular one or more substitutions selected fromthe group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W andR437W.

Embodiment 217

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 360, in particularthe substitution Q360S, in combination with an alteration at two or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 416, 437 and450, in particular two or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, E185P, I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y,F279W, H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, D416V, R437F,R437H, R437Y, R437W, H450F, H450Y and H450W, preferably two or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, D416V, R437W and H450W.

Embodiment 218

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 360, in particularthe substitution Q360S, in combination with a substitution at two ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 416 and 437, in particular two or more substitutions selected fromthe group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T, D416Vand R437W.

Embodiment 219

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 360, in particularthe substitution Q360S, in combination with a substitution at two ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 279 and 437, in particular two or more substitutions selected fromthe group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W andR437W.

Embodiment 220

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 416, in particularthe substitution D416V, in combination with an alteration at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 437 and450, in particular one or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, E185P, I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y,F279W, H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, R437F,R437H, R437Y, R437W, H450F, H450Y and H450W, preferably one or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, R437W and H450W.

Embodiment 221

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 416, in particularthe substitution D416V, in combination with a substitution at one ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 360 and 437, in particular one or more substitutions selected fromthe group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T, Q360Sand R437W.

Embodiment 222

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 416, in particularthe substitution D416V, in combination with a substitution at one ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 279 and 437, in particular one or more substitutions selected fromthe group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W andR437W.

Embodiment 223

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 416, in particularthe substitution D416V, in combination with an alteration at two or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 437 and450, in particular two or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, E185P, I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y,F279W, H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, R437F,R437H, R437Y, R437W, H450F, H450Y and H450W, preferably two or morealterations selected from the group consisting of A1AH, A1AW, A1H, A1W,N2NH, N2NW, N2H, N2W, H68W, G71W, N126W, H133Y, H142W, P144W, Y156W,Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W,L318W, Q360S, R437W and H450W.

Embodiment 224

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 416, in particularthe substitution D416V, in combination with a substitution at two ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 360 and 437, in particular two or more substitutions selected fromthe group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T, Q360Sand R437W.

Embodiment 225

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 416, in particularthe substitution D416V, in combination with a substitution at two ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158, 279 and 437, in particular two or more substitutions selected fromthe group consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W, F279W andR437W.

Embodiment 226

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 437, in particularthe substitution R437W, in combination with an alteration at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416 and450, in particular one or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, E185P, I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y,F279W, H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V,H450F, H450Y and H450W, preferably one or more alterations selected fromthe group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V andH450W.

Embodiment 227

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 437, in particularthe substitution R437W, in combination with a substitution at one ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 360 and 416, in particular one or more substitutions selected fromthe group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T, Q360Sand D416V.

Embodiment 228

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 437, in particularthe substitution R437W, in combination with a substitution at one ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158 and 279, in particular one or more substitutions selected from thegroup consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W and F279W.

Embodiment 229

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 437, in particularthe substitution R437W, in combination with an alteration at two or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416 and450, in particular two or more alterations selected from the groupconsisting of A1AH, A1AF, A1AY, A1AW, A1H, A1F, A1Y, A1W, N2NH, N2N2F,N2NY, N2NW, N2H, N2F, N2Y, N2W, H68F, H68Y, H68W, G71F, G71H, G71Y,G71W, N126F, N126H, N126Y, N126W, H133F, H133Y, H133W, H142F, H142Y,H142W, P144F, P144H, P144Y, P144W, Y156F, Y156H, Y156W, Y158F, Y158H,Y158W, K176L, E185P, I201F, I201Y, H205Y, K213T, S239A, S239Q, F279Y,F279W, H316F, H316Y, H316W, L318F, L318H, L318Y, L318W, Q360S, D416V,H450F, H450Y and H450W, preferably two or more alterations selected fromthe group consisting of A1AH, A1AW, A1H, A1W, N2NH, N2NW, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V andH450W.

Embodiment 230

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 437, in particularthe substitution R437W, in combination with a substitution at two ormore positions corresponding to any of positions 68, 176, 185, 201, 205,213, 360 and 416, in particular two or more substitutions selected fromthe group consisting of H68W, K176L, E185P, I201Y, H205Y, K213T, Q360Sand D416V.

Embodiment 231

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 437, in particularthe substitution R437W, in combination with a substitution at two ormore positions corresponding to any of positions 2, 68, 133, 142, 156,158 and 279, in particular two or more substitutions selected from thegroup consisting of N2H, H68W, H133Y, H142W, Y156W, Y158W and F279W.

Embodiment 232

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 450, in particularthe substitution H450W, in combination with a substitution at one ormore positions corresponding to any of positions 1, 2, 68, 71, 126, 133,142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360,416 and 437, in particular one or more substitutions selected from thegroup consisting of A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W,P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q,F279W, H316W, L318W, Q360S, D416V and R437W.

Embodiment 233

The variant of any of Embodiments 1 to 37, which comprises asubstitution at a position corresponding to position 450, in particularthe substitution H450W, in combination with a substitution at two ormore positions corresponding to any of positions 1, 2, 68, 71, 126, 133,142, 144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360,416 and 437, in particular two or more substitutions selected from thegroup consisting of A1H, A1W, N2H, N2W, H68W, G71W, N126W, H133Y, H142W,P144W, Y156W, Y158W, K176L, E185P, I201Y, H205Y, K213T, S239A, S239Q,F279W, H316W, L318W, Q360S, D416V and R437W.

Embodiment 234

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and K176L in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, I201Y, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of E185P, I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 235

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and K176L in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, I201Y, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of E185P, I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 236

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and E185P in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, I201Y, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of K176L, I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 237

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and E185P in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, I201Y, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of K176L, I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 238

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and I201Y in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of K176L, E185P, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 239

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and I201Y in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of K176L, E185P, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 240

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and H205Y in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of K176L, E185P, I201Y, K213T, Q360S, D416V and R437W.

Embodiment 241

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and H205Y in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of K176L, E185P, I201Y, K213T, Q360S, D416V and R437W.

Embodiment 242

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and K213T in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y,H205Y, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of K176L, E185P, I201Y, H205Y, Q360S, D416V and R437W.

Embodiment 243

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and K213T in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y,H205Y, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of K176L, E185P, I201Y, H205Y, Q360S, D416V and R437W.

Embodiment 244

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and Q360S in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of K176L, E185P, I201Y, H205Y, K213T, D416V and R437W.

Embodiment 245

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and Q360S in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of K176L, E185P, I201Y, H205Y, K213T, D416V and R437W.

Embodiment 246

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and D416V in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of K176L, E185P, I201Y, H205Y, K213T, Q360S and R437W.

Embodiment 247

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and D416V in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of K176L, E185P, I201Y, H205Y, K213T, Q360S and R437W.

Embodiment 248

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and R437W in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V and H450W,in particular one or more substitutions selected from the groupconsisting of K176L, E185P, I201Y, H205Y, K213T, Q360S and D416V.

Embodiment 249

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H68W and R437W in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V and H450W,in particular two or more substitutions selected from the groupconsisting of K176L, E185P, I201Y, H205Y, K213T, Q360S and D416V.

Embodiment 250

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K176L and E185P in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, I201Y, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 251

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K176L and E185P in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, I201Y, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, I201Y, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 252

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K176L and I201Y in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, E185P, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 253

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K176L and I201Y in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, E185P, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 254

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K176L and H205Y in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, I201Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, E185P, I201Y, K213T, Q360S, D416V and R437W.

Embodiment 255

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K176L and H205Y in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, I201Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, E185P, I201Y, K213T, Q360S, D416V and R437W.

Embodiment 256

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K176L and K213T in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, I201Y,H205Y, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, E185P, I201Y, H205Y, Q360S, D416V and R437W.

Embodiment 257

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K176L and K213T in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, I201Y,H205Y, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, E185P, I201Y, H205Y, Q360S, D416V and R437W.

Embodiment 258

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K176L and Q360S in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, E185P, I201Y, H205Y, K213T, D416V and R437W.

Embodiment 259

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K176L and Q360S in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, E185P, I201Y, H205Y, K213T, D416V and R437W.

Embodiment 260

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K176L and D416V in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, E185P, I201Y, H205Y, K213T, Q360S and R437W.

Embodiment 261

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K176L and D416V in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, E185P, I201Y, H205Y, K213T, Q360S and R437W.

Embodiment 262

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K176L and R437W in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V and H450W,in particular one or more substitutions selected from the groupconsisting of K176L, E185P, I201Y, H205Y, K213T, Q360S and D416V.

Embodiment 263

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K176L and R437W in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, E185P, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V and H450W,in particular two or more substitutions selected from the groupconsisting of K176L, E185P, I201Y, H205Y, K213T, Q360S and D416V.

Embodiment 264

The variant of any of embodiments 1 to 37 which comprises thesubstitutions E185P and I201Y in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 265

The variant of any of embodiments 1 to 37 which comprises thesubstitutions E185P and I201Y in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, H205Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, H205Y, K213T, Q360S, D416V and R437W.

Embodiment 266

The variant of any of embodiments 1 to 37 which comprises thesubstitutions E185P and H205Y in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, I201Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, I201Y, K213T, Q360S, D416V and R437W.

Embodiment 267

The variant of any of embodiments 1 to 37 which comprises thesubstitutions E185P and H205Y in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, I201Y,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, I201Y, K213T, Q360S, D416V and R437W.

Embodiment 268

The variant of any of embodiments 1 to 37 which comprises thesubstitutions E185P and K213T in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, I201Y,H205Y, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, I201Y, H205Y, Q360S, D416V and R437W.

Embodiment 269

The variant of any of embodiments 1 to 37 which comprises thesubstitutions E185P and K213T in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, I201Y,H205Y, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, I201Y, H205Y, Q360S, D416V and R437W.

Embodiment 270

The variant of any of embodiments 1 to 37 which comprises thesubstitutions E185P and Q360S in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, I201Y, H205Y, K213T, D416V and R437W.

Embodiment 271

The variant of any of embodiments 1 to 37 which comprises thesubstitutions E185P and Q360S in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, I201Y, H205Y, K213T, D416V and R437W.

Embodiment 272

The variant of any of embodiments 1 to 37 which comprises thesubstitutions E185P and D416V in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, I201Y, H205Y, K213T, Q360S and R437W.

Embodiment 273

The variant of any of embodiments 1 to 37 which comprises thesubstitutions E185P and D416V in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, I201Y, H205Y, K213T, Q360S and R437W.

Embodiment 274

The variant of any of embodiments 1 to 37 which comprises thesubstitutions E185P and R437W in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, I201Y, H205Y, K213T, Q360S and D416V.

Embodiment 275

The variant of any of embodiments 1 to 37 which comprises thesubstitutions E185P and R437W in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, I201Y,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, I201Y, H205Y, K213T, Q360S and D416V.

Embodiment 276

The variant of any of embodiments 1 to 37 which comprises thesubstitutions I201Y and H205Y in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, K213T, Q360S, D416V and R437W.

Embodiment 277

The variant of any of embodiments 1 to 37 which comprises thesubstitutions I201Y and H205Y in combination with or more substitutionsselected from the group consisting of A1H, A1W, N2H, N2W, H68W, G71W,N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P, K213T, S239A,S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W, in particulartwo or more substitutions selected from the group consisting of H68W,K176L, E185P, K213T, Q360S, D416V and R437W.

Embodiment 278

The variant of any of embodiments 1 to 37 which comprises thesubstitutions I201Y and K213T in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,H205Y, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, H205Y, Q360S, D416V and R437W.

Embodiment 279

The variant of any of embodiments 1 to 37 which comprises thesubstitutions I201Y and K213T in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,H205Y, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, E185P, H205Y, Q360S, D416V and R437W.

Embodiment 280

The variant of any of embodiments 1 to 37 which comprises thesubstitutions I201Y and Q360S in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, H205Y, K213T, D416V and R437W.

Embodiment 281

The variant of any of embodiments 1 to 37 which comprises thesubstitutions I201Y and Q360S in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, E185P, H205Y, K213T, D416V and R437W.

Embodiment 282

The variant of any of embodiments 1 to 37 which comprises thesubstitutions I201Y and D416V in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, H205Y, K213T, Q360S and R437W.

Embodiment 283

The variant of any of embodiments 1 to 37 which comprises thesubstitutions I201Y and D416V in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, E185P, H205Y, K213T, Q360S and R437W.

Embodiment 284

The variant of any of embodiments 1 to 37 which comprises thesubstitutions I201Y and R437W in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, H205Y, K213T, Q360S and D416V.

Embodiment 285

The variant of any of embodiments 1 to 37 which comprises thesubstitutions I201Y and R437W in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, E185P, H205Y, K213T, Q360S and D416V.

Embodiment 286

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H205Y and K213T in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, Q360S, D416V and R437W.

Embodiment 287

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H205Y and K213T in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, Q360S, D416V and R437W.

Embodiment 288

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H205Y and Q360S in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, S239A, S239Q, F279W, H316W, L318W, D416V, R437W and H450W, inparticular one or more substitutions selected from the group consistingof H68W, K176L, E185P, I201Y, K213T, D416V and R437W.

Embodiment 289

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H205Y and Q360S in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, S239A, S239Q, F279W, H316W, L318W, D416V, R437W and H450W, inparticular two or more substitutions selected from the group consistingof H68W, K176L, E185P, I201Y, K213T, D416V and R437W.

Embodiment 290

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H205Y and D416V in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, K213T, Q360S and R437W.

Embodiment 291

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H205Y and D416V in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, K213T, Q360S and R437W.

Embodiment 292

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H205Y and R437W in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, K213T, Q360S and D416V.

Embodiment 293

The variant of any of embodiments 1 to 37 which comprises thesubstitutions H205Y and R437W in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, K213T, Q360S and D416V.

Embodiment 294

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K213T and Q360S in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, S239A, S239Q, F279W, H316W, L318W, D416V, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, H205Y, D416V and R437W.

Embodiment 295

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K213T and Q360S in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, S239A, S239Q, F279W, H316W, L318W, D416V, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, H205Y, D416V and R437W.

Embodiment 296

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K213T and D416V in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, S239A, S239Q, F279W, H316W, L318W, Q360S, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, H205Y, Q360S and R437W.

Embodiment 297

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K213T and D416V in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, S239A, S239Q, F279W, H316W, L318W, Q360S, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, H205Y, Q360S and R437W.

Embodiment 298

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K213T and R437W in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, H205Y, Q360S and D416V.

Embodiment 299

The variant of any of embodiments 1 to 37 which comprises thesubstitutions K213T and R437W in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, S239A, S239Q, F279W, H316W, L318W, Q360S, D416V and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, H205Y, Q360S and D416V.

Embodiment 300

The variant of any of embodiments 1 to 37 which comprises thesubstitutions Q360S and D416V in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, R437W and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, H205Y, K213T and R437W.

Embodiment 301

The variant of any of embodiments 1 to 37 which comprises thesubstitutions Q360S and D416V in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, R437W and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, H205Y, K213T and R437W.

Embodiment 302

The variant of any of embodiments 1 to 37 which comprises thesubstitutions Q360S and R437W in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, D416V and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, H205Y, K213T and D416V.

Embodiment 303

The variant of any of embodiments 1 to 37 which comprises thesubstitutions Q360S and R437W in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, D416V and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, H205Y, K213T and D416V.

Embodiment 304

The variant of any of embodiments 1 to 37 which comprises thesubstitutions D416V and R437W in combination with one or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S and H450W,in particular one or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, H205Y, K213T and Q360S.

Embodiment 305

The variant of any of embodiments 1 to 37 which comprises thesubstitutions D416V and R437W in combination with two or moresubstitutions selected from the group consisting of A1H, A1W, N2H, N2W,H68W, G71W, N126W, H133Y, H142W, P144W, Y156W, Y158W, K176L, E185P,I201Y, H205Y, K213T, S239A, S239Q, F279W, H316W, L318W, Q360S and H450W,in particular two or more substitutions selected from the groupconsisting of H68W, K176L, E185P, I201Y, H205Y, K213T and Q360S.

Embodiment 306

The variant of any of Embodiments 1-305, which further comprises asubstitution at one or more positions corresponding to any of positions15, 48, 49, 107, 138, 156, 181, 187, 188, 190, 197, 209, 239, 255, 264,299, 474 and 475 of SEQ ID NO: 1.

Embodiment 307

The variant of Embodiment 306, wherein the variant comprises:

-   -   a substitution at a position corresponding to position 15 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Leu, Ser or        Thr;    -   a substitution at a position corresponding to position 48 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ala;    -   a substitution at a position corresponding to position 49 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,        Phe, Pro, Ser, Trp, Tyr or Val, in particular with Gly, His, lie        or Leu;    -   a substitution at a position corresponding to position 107 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ala;    -   a substitution at a position corresponding to position 138 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,        Phe, Pro, Ser, Thr, Tyr or Val, in particular with Phe or Tyr;    -   a substitution at a position corresponding to position 156 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Tyr;    -   a substitution at a position corresponding to position 181 with        Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Asp, Glu or        Thr;    -   a substitution at a position corresponding to position 187 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,        Phe, Pro, Thr, Trp, Tyr or Val, in particular with Asp;    -   a substitution at a position corresponding to position 188 with        Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ser or Thr;    -   a substitution at a position corresponding to position 190 with        Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Phe;    -   a substitution at a position corresponding to position 197 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with lie, Leu,        Ser, Thr or Val;    -   a substitution at a position corresponding to position 209 with        Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Val;    -   a substitution at a position corresponding to position 239 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,        Phe, Pro, Thr, Trp, Tyr or Val, in particular with Ala, Asn,        Asp, Gln, Glu, Met or Cys;    -   a substitution at a position corresponding to position 255 with        Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Gly or Pro;    -   a substitution at a position corresponding to position 264 with        Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Ser;    -   a substitution at a position corresponding to position 299 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Arg;    -   a substitution at a position corresponding to position 474 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Arg, Gln, Glu        or Lys; or    -   a substitution at a position corresponding to position 475 with        Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe,        Pro, Ser, Thr, Trp, Tyr or Val, in particular with Arg, Gln, Glu        or Lys.

Embodiment 308

The variant of any of Embodiments 306 to 307, which comprises one ormore substitutions selected from the group consisting of M15T, M15S,M15L, G48A, T49L, T49I, G107A, W138Y, W138F, H156Y, A181T, A181E, A181D,S187D, N188S, N188T, N190F, M197S, M197T, M197V, M197L, M1971, A209V,S239Q, S239E, S239N, S239D, S239A, S239M, S239C, E255P, E255G, Q264S,G299R, G474K, G474R, G474E, G474Q, G475K, G475R, G475E and G475Q.

Embodiment 309

The variant of any of Embodiments 1 to 308, which comprises or consistsof a set of substitutions selected from the group consisting of:

A1H+N2W+K176L+E185P,

A1W+N2H+K176L+E185P,

N2H+H68W+H133Y+K176L+E185P,

N2H+H68W+Y156W+K176L+E185P,

N2H+H68W+Y158W+K176L+E185P,

N2H+H68W+K176L+E185P,

N2H+H68W+K176L+E185P+I201Y+H205Y+D207V+V209D,

N2H+H68W+K176L+E185P+F279W,

N2H+H133Y+K176L+E185P+H316W+R437W,

N2H+H133Y+K176L+E185P+Q360S+R437W,

N2H+H142W+K176L+E185P+H316W+R437W,

N2H+H142W+K176L+E185P+Q360S+R437W,

N2H+P144W+K176L+E185P,

N2H+Y156W+Y158W+K176L+E185P+H316W+R437W,

N2H+Y156W+K176L+E185P+Q360S+R437W,

N2H+Y158W+K176L+E185P+I201Y+H205Y+D207V+V209D+H316W,

N2H+K176L+E185P,

N2H+K176L+E185P+H316W,

N2H+K176L+E185P+H316W+L318W+R437W,

N2H+K176L+E185P+H316W+Q360S+R437W,

N2H+K176L+E185P+H316W+R437W,

N2H+K176L+E185P+R437W,

N2H+K176L+E185P+Q360S+R437W,

N2H+K176L+E185P+H316W+Q360S+R437W,

N2H+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H68W+K176L+E185P,

H68W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H68W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

G71W+K176L+E185P,

N126W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H133Y+Y158W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H133Y+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H142W+Y158W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H142W+K176L+E185P,

H142W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

H142W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

P144W+K176L+E185P,

Y156W+Y158W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

Y156W+Y158W+K176L+E185P+H316W+R437W,

Y156W+K176L+E185P+Q360S+R437W,

Y156W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

Y158W+K176L+E185P,

Y158W+K176L+E185P+I201Y+H205Y+D207V+V209D+H316W,

Y158W+K176L+E185P+I201Y+H205Y+K213T+H316L+L318W+Q360S+D416V+R437W,

Y158W+K176L+E185P+I201Y+H205Y+K213T+H316W+Q360S+D416V+R437W,

Y158W+K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

Y158W+K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

K176L+E185P,

K176L+E185P+I201Y+H205Y+K213T+Q360S+D416V+R437W,

K176L+E185P+I201Y+H205Y+R437W,

K176L+E185P+F279W,

K176L+E185P+H316W,

K176L+E185P+L318W,

K176L+E185P+H450W,

K176L+I201Y+H205Y+K213T+S239Q+Q360S+D416V+R437W,

K176L+I201Y+H205Y+K213T+H316W+Q360S+D416V+R437W,

K176L+I201Y+H205Y+K213T+L318W+Q360S+D416V+R437W,

K176L+I201Y+H205Y+K213T+Q360S+D416V+R437W,

K176L+I201Y+H205Y+K213T+Q360S+R437W,

K176L+I201Y+H205Y+K213T+D416V+R437W, and

K176L+I201Y+H205Y+Q360S+D416V+R437W.

Embodiment 310

The variant of any of Embodiments 1 to 309 which further comprises adeletion at both of the two positions immediately before position 180 ofSEQ ID NO: 1.

Embodiment 311

The variant of any of Embodiments 1 to 310, wherein the total number ofsubstitutions is 2-20, e.g., 2-10 or 2-5, such as 2, 3, 4, 5, 6, 7, 8, 9or 10 substitutions.

Embodiment 312

The variant of any of Embodiments 1 to 311, which has increasedthermostability compared to the mature polypeptide of SEQ ID NO: 1 whenincubated at high temperature, low calcium and low pH in the presence ofat least 0.1% starch, e.g., in the presence of 0.9% or 1% starch.

Embodiment 313

The variant of any of Embodiments 1 to 311, which has increasedthermostability compared to the mature polypeptide of SEQ ID NO: 14 whenincubated at high temperature, low calcium and low pH in the presence ofat least 0.1% starch, e.g., in the presence of 0.9% or 1% starch.

Embodiment 314

The variant of any of Embodiments 1 to 311, which has increasedthermostability compared to its parent when incubated at hightemperature, low calcium and low pH in the presence of at least 0.1%starch, e.g., in the presence of 0.9% or 1% starch.

Embodiment 315

The variant of any of Embodiments 1 to 311, which has an increasedresidual activity half-life compared to the mature polypeptide of SEQ IDNO: 1 when incubated with 0.125 mM CaCl₂, 0.9% starch at 95° C. and pH4.8.

Embodiment 316

The variant of any of Embodiments 1 to 311, which has an increasedresidual activity half-life compared to the mature polypeptide of SEQ IDNO: 14 when incubated with 0.125 mM CaCl₂, 0.9% starch at 95° C. and pH4.8.

Embodiment 317

The variant of any of Embodiments 1 to 311, which has an increasedresidual activity half-life compared to its parent when incubated with0.125 mM CaCl₂, 0.9% starch at 95° C. and pH 4.8.

Embodiment 318

The variant of any of Embodiments 1 to 311, which has an increasedresidual activity half-life compared to the mature polypeptide of SEQ IDNO: 1 when incubated with 0.125 mM CaCl₂, 0.9% starch at 95° C. and pH4.5.

Embodiment 319

The variant of any of Embodiments 1 to 311, which has an increasedresidual activity half-life compared to the mature polypeptide of SEQ IDNO: 14 when incubated with 0.125 mM CaCl₂, 0.9% starch at 95° C. and pH4.5.

Embodiment 320

The variant of any of Embodiments 1 to 311, which has an increasedresidual activity half-life compared to its parent when incubated with0.125 mM CaCl₂, 0.9% starch at 95° C. and pH 4.5.

Embodiment 321

A detergent composition comprising the variant of any of Embodiments 1to 320 and a surfactant.

Embodiment 322

A composition comprising the variant of any of Embodiments 1 to 320 andone or more enzymes selected from the group consisting of beta-amylase,cellulase (beta-glucosidase, cellobiohydrolase, and endoglucanase)glucoamylase, hemicellulase (e.g., xylanase), isoamylase, isomerase,lipase, phytase, protease and pullulanase.

Embodiment 323

Use of the variant of any of Embodiments 1 to 320 for washing and/ordishwashing.

Embodiment 324

Use of the variant of any of Embodiments 1 to 320 for desizing atextile.

Embodiment 325

Use of the variant of any of Embodiments 1 to 320 for producing a bakedproduct

Embodiment 326

Use of the variant of any of Embodiments 1 to 320 for liquefying astarch-containing material.

Embodiment 327

A method of producing liquefied starch, comprising liquefying astarch-containing material with the variant of any of Embodiments 1 to320.

Embodiment 328

A method of producing a fermentation product, comprising

-   -   (a) liquefying a starch-containing material with the variant of        any of Embodiments 1 to 320 to produce a liquefied mash;    -   (b) saccharifying the liquefied mash to produce fermentable        sugars; and    -   (c) fermenting the fermentable sugars in the presence of a        fermenting organism.

Embodiment 329

The method of Embodiment 328, wherein step (a) is performed at pH 4-5.

Embodiment 330

A method of producing a fermentation product, comprising contacting astarch substrate with the variant of any of Embodiments 1 to 320, aglucoamylase, and a fermenting organism.

Embodiment 331

An isolated polynucleotide encoding the variant of any of Embodiments 1to 320.

Embodiment 332

A nucleic acid construct comprising the polynucleotide of Embodiment331.

Embodiment 333

An expression vector comprising the polynucleotide of Embodiment 331.

Embodiment 334

A host cell comprising the polynucleotide of Embodiment 331.

Embodiment 335

A method of producing an alpha-amylase variant, comprising:

-   -   a. cultivating the host cell of Embodiment 334 under conditions        suitable for expression of the variant; and    -   b. recovering the variant.

Embodiment 336

A method for obtaining an alpha-amylase variant, comprising (a)introducing into a parent alpha-amylase a substitution at one or morepositions corresponding to any of positions 1, 2, 68, 71, 126, 133, 142,144, 156, 158, 176, 185, 201, 205, 213, 239, 279, 316, 318, 360, 416,437 and 450 of SEQ ID NO: 1; and (b) recovering the variant.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Assays for Measurement of Amylolytic Activity (Alpha-AmylaseActivity)

PNP-G7 Assay:

The alpha-amylase activity is determined by a method employing thePNP-G7 substrate. PNP-G7 is an abbreviation for4,6-ethylidene(G₇)-p-nitrophenyl(G₁)-α,D-maltoheptaoside, a blockedoligosaccharide which can be cleaved by an endo-amylase, such as analpha-amylase. Following the cleavage, the alpha-glucosidase included inthe kit digest the hydrolysed substrate further to liberate a free PNPmolecule which has a yellow color and thus can be measured by visiblespectophometry at λ=405 nm (400-420 nm.). Kits containing PNP-G7substrate and alpha-glucosidase is manufactured by Roche/Hitachi (cat.No. 11876473).

Reagents:

The G7-PNP substrate from this kit contains 22 mM 4,6-ethylidene-G7-PNPand 52.4 mM HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonicacid), pH 7.0.

The alpha-glucosidase reagent contains 52.4 mM HEPES, 87 mM NaCl, 12.6mM MgCl₂, 0.075 mM CaCl₂, >4 kU/L alpha-glucosidase.

The substrate working solution is made by mixing 1 ml of thealpha-glucosidase reagent with 0.2 ml of the G7-PNP substrate. Thissubstrate working solution is made immediately before use.

Dilution buffer: 50 mM EPPS, 0.01% (w/v) Triton X100 (polyethyleneglycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (C₁₄H₂₂O(C₂H₄O)_(n)(n=9-10))), 1 mM CaCl₂, pH 7.0.

Procedure:

The amylase sample to be analyzed is diluted in dilution buffer toensure the pH in the diluted sample is 7. The assay is performed bytransferring 20 μl diluted enzyme samples to 96 well microtiter plateand adding 80 μl substrate working solution. The solution is mixed andpre-incubated 1 minute at room temperature and absorption is measuredevery 20 sec. over 5 minutes at OD 405 nm.

The slope (absorbance per minute) of the time dependent absorption-curveis directly proportional to the specific activity (activity per mgenzyme) of the alpha-amylase in question under the given set ofconditions. The amylase sample should be diluted to a level where theslope is below 0.4 absorbance units per minute.

Phadebas Activity Assay:

The alpha-amylase activity can also be determined by a method using thePhadebas substrate (from for example Magle Life Sciences, Lund, Sweden).A Phadebas tablet includes interlinked starch polymers that are in theform of globular microspheres that are insoluble in water. A blue dye iscovantly bound to these microspheres. The interlinked starch polymers inthe microsphere are degraded at a speed that is proportional to thealpha-amylase activity. When the alpha-amylse degrades the starchpolymers, the released blue dye is water soluble and concentration ofdye can be determined by measuring absorbance at 620 nm. Theconcentration of blue is proportional to the alpha-amylase activity inthe sample.

The amylase sample to be analysed is diluted in dilution buffer with thedesired pH. One substrate tablet is suspended in 5 mL activity bufferand mixed on magnetic stirrer. During mixing of substrate transfer 150μl to microtiter plate (MTP) or PCR-MTP. Add 30 μl diluted amylasesample to 150 μl substrate and mix. Incubate for 15 minutes at 37° C.The reaction is stopped by adding 30 μl 1M NaOH and mix. Centrifuge MTPfor 5 minutes at 4000×g. Transfer 100 μl to new MTP and measureabsorbance at 620 nm.The amylase sample should be diluted so that the absorbance at 620 nm isbetween 0 and 2.2.EnzChek® Assay:

For the determination of residual amylase activity an EnzChek® UltraAmylase Assay Kit (E33651, Invitrogen, La Jolla, Calif., USA) was used.

The substrate is a corn starch derivative, DQ™ starch, which is cornstarch labeled with BODIPY® FL dye to such a degree that fluorescence isquenched. One vial containing approx. 1 mg lyophilized substrate isdissolved in 100 microliters of 50 mM sodium acetate (pH 4.0). The vialis vortexed for 20 seconds and left at room temperature, in the dark,with occasional mixing until dissolved. Then 900 microliters of 100 mMacetate, 0.01% (w/v) TRITON® X100, 0.125 mM CaCl₂, pH 5.5 is added,vortexed thoroughly and stored at room temperature, in the dark untilready to use. The stock substrate working solution is prepared bydiluting 10-fold in residual activity buffer (100 mM acetate, 0.01%(w/v) TRITON® X100, 0.125 mM CaCl₂, pH 5.5). Immediately afterincubation the enzyme is diluted to a concentration of 10-20 ng enzymeprotein/ml in 100 mM acetate, 0.01% (W/v) TRITON® X100, 0.125 mM CaCl₂,pH 5.5.

For the assay, 25 microliters of the substrate working solution is mixedfor 10 second with 25 microliters of the diluted enzyme in a black 384well microtiter plate. The fluorescence intensity is measured(excitation: 485 nm, emission: 555 nm) once every minute for 15 minutesin each well at 25° C. and the V_(max) is calculated as the slope of theplot of fluorescence intensity against time. The plot should be linearand the residual activity assay has been adjusted so that the dilutedreference enzyme solution is within the linear range of the activityassay.

Reference Alpha-Amylase and Variants Thereof

The reference alpha-amylase is LE399 (previously disclosed in, e.g., WO2002/010355). LE399 comprises amino acids 1-37 of the alpha-amylase fromBacillus amyloliquefaciens (SEQ ID NO: 6) and amino acids 40-483 of thealpha-amylase from Bacillus licheniformis (SEQ ID NO: 1) with thefollowing substitutions G48A T49I G107A H156Y A181T N190F I201F A209VQ264S.

The variants tested are variants of LE399, so the substitutions in eachvariant as listed in the tables below are substitutions as compared toLE399. The position numbering is according to SEQ ID NO: 1.

LE399 is two amino acids shorter than SEQ ID NO: 1 in the N-terminal,i.e. there are no amino acids corresponding to positions 1 and 2 of SEQID NO: 1 in LE399. The alteration denoted in the tables as *2aH meansinsertion of H before the N-terminal V of LE399. A similar alteration inSEQ ID NO: 1 would be substitution of amino acid N2 with H, i.e. N2H(alternatively, deletion of amino acid A1 combined with substitution ofamino acid N2 with H, i.e. A1* N2H). Likewise, the alterations denotedin the tables as *2aH *2bW means insertion of HW before the N-terminal Vof LE399. A similar alteration in SEQ ID NO: 1 would be thesubstitutions A1H N2W.

Example 1 Thermostability of Alpha-Amylase Variants with 0.9% Starch atpH 4.5

The thermostability of a reference alpha-amylase and alpha-amylasevariants thereof was determined by incubating the referencealpha-amylase and variants at pH 4.5 and 90 or 95° C. with 0.125 mMCaCl₂ and 0.9% starch (corn starch) followed by residual activitydetermination using the EnzChek® substrate (EnzChek® Ultra Amylase assaykit, E33651, Molecular Probes).

Purified enzyme samples were diluted to working concentrations of 5 and10 ppm (micrograms/ml) in enzyme dilution buffer (10 mM acetate, 0.01%Triton X100, 0.125 mM CaCl₂, pH 5.0). Twenty microliters enzyme samplewas transferred to 96-well PCR MTP and 180 microliters stability buffer(150 mM acetate, 0.01% Triton X100, 0.125 mM CaCl₂, pH 4.5, +/−0.9%starch) was added to each well and mixed. The assay was performed usingtwo concentrations of enzyme in duplicates. Before incubation at 90 or95° C., 20 microliters was withdrawn and stored on ice as unstressedcontrol samples. Incubation was performed in a PCR machine (pH 4.5,90/95° C., 0.125 mM CaCl₂, +/−0.9% starch). Incubation time (minutes)was selected so that residual activity was between 6-84%. If theresidual activity is outside this interval then it is indicated by > or<.

After incubation samples were diluted to 10-20 ng enzyme protein/ml inresidual activity buffer (100 mM Acetate, 0.01% Triton X100, 0.125 mMCaCl₂, pH 5.5) and 25 microliters diluted enzyme was transferred toblack 384-well MTP. Residual activity was determined using the EnzChek®substrate by adding 25 microliters substrate solution (100micrograms/ml) to each well. Fluorescence was determined at 25° C. everyminute for 15 minutes using excitation filter at 485-P nm and emissionfilter at 555 nm (fluorescence reader is Polarstar, BMG). The residualactivity was normalized to unstressed control samples for each setup.

Assuming exponential decay half life time (T½ (min)) was calculatedusing the equation: T½ (min)=T(min)*LN(0.5)/LN(% RA/100), where T isassay incubation time in minutes, and % RA is % residual activitydetermined in assay.

Using this assay setup the half life time was determined as a measure ofthermostability for the reference alpha-amylase and variants thereof asshown in Tables 1 and 2.

Reference amylase was incubated at pH 4.5, 0.125 mM CaCl₂, +/−0.9%starch, at 90° C. for 5 minutes (Table 1).

TABLE 1 pH 4.5, 0.125 mM CaCl₂, 90° C. for 5 or 15 minutes in 0.9%starch pH 4.5, 0.125 mM CaCl₂, 90° C. in buffer for 5 or 8 minutes T½(min) T½ (min) (pH 4.5, 0.125 mM (pH 4.5, 0.125 mM CaCl₂, buffer, CaCl₂,0.9% Mutations 90° C.) starch, 90° C.) Reference 2 3 K176L F201Y H205YK213T Q360S D416V R437W 6 11 K176L F201Y H205Y K213T Q360S D416V R437WH133Y 14 17 K176L F201Y H205Y K213T Q360S D416V R437W S239Q 18 21 K176LF201Y H205Y K213T Q360S D416V R437W H316W 5 10 K176L F201Y H205Y K213TQ360S D416V R437W L318W 6 10 K176L F201Y H205Y K213T Q360S D416V R437W*2aH 6 10 K176L F201Y H205Y K213T Q360S D416V R437W N126W 2 5 K176LF201Y H205Y K213T Q360S D416V R437W H68W 5 11 K176L F201Y H205Y K213TQ360S D416V R437W Y158W 6 11 K176L F201Y H205Y K213T Q360S D416V R437WY156W 5 11 K176L F201Y H205Y K213T Q360S D416V R437W H142W 7 11

TABLE 2 pH 4.5, 0.125 mM CaCl₂, 95° C. in 0.9% starch for 30 or 50minutes pH 4.5, 0.125 mM CaCl₂, 95° C. in buffer for 30 minutes T½ (min)T½ (min) (pH 4.5 0.125 mM (pH 4.5 0.125 mM CaCl₂ buffer CaCl₂ 0.9%Mutations 95° C.) starch 95° C.) K176L E185P 20 27 *2aH K176L E185P 1837 *2aH *2bW K176L E185P 21 30 H68W K176L E185P 17 33 G71W K176L E185P20 32 P144W K176L E185P 18 36 Y158W K176L E185P 17 28 K176L E185P F279W20 33 K176L E185P H316W 23 38 K176L E185P *2aH R437W 23 >120 K176L E185PH450W 20 46 K176L E185P *2aH H316W 22 65 *2aH P144W K176L E185P 19 71K176L F201Y H205Y K213T Q360S D416V R437W H133Y <0 32 K176L F201Y H205YK213T Q360S D416V R437W <0 14 H142W K176L E185P 13 43 K176L E185P L318W12 41 *2aH Y158W K176L E185P F201Y H205Y D207V V209D H316W 24 63 *2aHK176L E185P 18 36 *2aH H68W K176L E185P H133Y 27 74 *2aH K176L E185PH316W R437W 24 44 *2aH K176L E185P H316W R437W L318W 20 37 *2aH K176LE185P H316W R437W H133Y 32 60 *2aH K176L E185P H316W R437W Y156W Y158W22 44 *2aH K176L E185P H316W R437W H142W 23 59 *2aH K176L E185P R437WQ360S 20 36 *2aH K176L E185P R437W Q360S Y156W 18 34 *2aH K176L E185PR437W Q360S H142W 16 27 *2aH K176L E185P R437W Q360S H133Y 26 42 *2aHK176L E185P R437W Q360S H316W 23 38 *2aH K176L E185P H316W R437W Q360S22 34 K176L F201Y H205Y K213T Q360S D416V R437W E185P 19 30 K176L F201YH205Y K213T Q360S D416V R437W E185P H142W 18 25 K176L F201Y H205Y K213TQ360S D416V R437W E185P H68W 18 25 K176L F201Y H205Y K213T Q360S D416VR437W E185P Y158W H133Y 24 33 K176L F201Y H205Y K213T Q360S D416V R437WE185P Y158W H142W 19 31 K176L F201Y H205Y K213T Q360S D416V R437W E185PY158W Y156W 21 28 K176L F201Y H205Y K213T Q360S D416V R437W E185P Y158WH316W 24 32 K176L F201Y H205Y K213T Q360S D416V R437W E185P Y158W H316LL318W 18 20 *2aH H68W K176L E185P 15 39 *2aH H68W K176L E185P Y158W 1730 *2aH H68W K176L E185P Y156W 16 28 *2aH H68W K176L E185P F279W 14 44*2aH H68W K176L E185P F201Y H205Y D207V V209D 14 16 *2aH H68W K176LE185P H133Y 23 78

Example 2 Thermostability of Alpha-Amylase Variants with 0.9% Starch atpH 4.8

The thermostability of a reference alpha-amylase and alpha-amylasevariants thereof was determined by incubating the referencealpha-amylase and variants at pH 4.8 and 90 or 95° C. with 0.125 mMCaCl₂+/−0.9% starch (corn starch) followed by residual activitydetermination using the EnzChek® substrate (EnzChek® Ultra Amylase assaykit, E33651, Molecular Probes).

Purified enzyme samples were diluted to working concentrations of 5 and10 ppm (micrograms/ml) in enzyme dilution buffer (10 mM acetate, 0.01%Triton X100, 0.125 mM CaCl₂, pH 5.0). Twenty microliters enzyme samplewas transferred to 96-well PCR MTP and 180 microliters stability buffer(150 mM acetate, 0.01% Triton X100, 0.125 mM CaCl₂, pH 4.8, +/−0.9%starch) was added to each well and mixed. The assay was performed usingtwo concentrations of enzyme in duplicates. Before incubation at 90 or95° C., 20 microliters was withdrawn and stored on ice as unstressedcontrol samples. Incubation was performed in a PCR machine (pH 4.8,90/95° C., 0.125 mM CaCl₂, +/−0.9% starch). Incubation time (minutes)was selected so that residual activity was between 6-84%. If theresidual activity is outside this interval then it is indicated by > or<.

After incubation samples were diluted to 10-20 ng enzyme protein/ml inresidual activity buffer (100 mM Acetate, 0.01% Triton X100, 0.125 mMCaCl₂, pH 5.5) and 25 microliters diluted enzyme was transferred toblack 384-well MTP. Residual activity was determined using the EnzChek®substrate by adding 25 microliters substrate solution (100micrograms/ml) to each well. Fluorescence was determined at 25° C. everyminute for 15 minutes using excitation filter at 485-P nm and emissionfilter at 555 nm (fluorescence reader is Polarstar, BMG). The residualactivity was normalized to unstressed control samples for each setup.

Assuming exponential decay half life time (T½ (min)) was calculatedusing the equation: T½ (min)=T(min)*LN(0.5)/LN(% RA/100), where T isassay incubation time in minutes, and % RA is % residual activitydetermined in assay.

Using this assay setup the half life time was determined as a measure ofthermostability for the reference alpha-amylase and variants thereof asshown in Tables 3-5.

TABLE 3 Conditions: pH 4.8, 0.125 mM CaCl₂, 95° C. in 0.9% starch for 8minutes pH 4.8, 0.125 mM CaCl₂, 90° C. in buffer for 12 minutes T½ (min)T½ (min) (pH 4.8, 0.125 mM (pH 4.8, 0.125 mM CaCl₂, buffer, CaCl₂, 0.9%Mutations 90° C.) starch, 95° C.) Reference 10 4

TABLE 4 Conditions: pH 4.8, 0.125 mM CaCl₂, 90° C. in 0.9% starch for 90minutes pH 4.8, 0.125 mM CaCl₂, 90° C. in buffer for 40 minutes T½ (min)T½ (min) (pH 4.8 0.125 mM (pH 4.8 0.125 mM CaCl₂ buffer CaCl₂ 0.9%Mutations 90° C.) starch 90° C.) K176L F201Y H205Y K213T Q360S D416VR437W 23 58 K176L F201Y H205Y K213T Q360S D416V R437W H133Y 84 122 K176LF201Y H205Y K213T Q360S D416V R437W S239Q 56 153 K176L F201Y H205Y K213TQ360S D416V R437W H316W 19 51 K176L F201Y H205Y K213T Q360S D416V R437WL318W 25 58 K176L F201Y H205Y K213T Q360S D416V R437W *2aH 22 58 K176LF201Y H205Y K213T Q360S D416V R437W N126W 11 43 K176L F201Y H205Y K213TQ360S D416V R437W H68W 22 24 K176L F201Y H205Y K213T Q360S D416V R437WY158W 29 68 K176L F201Y H205Y K213T Q360S D416V R437W Y156W 20 54 K176LF201Y H205Y K213T Q360S D416V R437W H142W 26 57

TABLE 5 Conditions: pH 4.8, 0.125 mM CaCl₂, 95° C. in 0.9% starch for40, 70 or 90 minutes pH 4.8, 0.125 mM CaCl₂, 95° C. in buffer for 15 or50 minutes T½ (min) T½ (min) (pH 4.8 0.125 mM (pH 4.8 0.125 mM CaCl₂buffer CaCl₂ 0.9% Mutations 95° C.) starch 95° C.) K176L E185P 42 73*2aH K176L E185P 42 127 *2aH *2bW K176L E185P 39 79 H68W K176L E185P 4393 G71W K176L E185P 43 95 H142W K176L E185P 32 82 P144W K176L E185P 4870 Y158W K176L E185P 36 97 K176L E185P F279W 41 110 K176L E185P H316W 4391 K176L E185P L318W 34 78 K176L E185P *2aH R437W 44 209 K176L F201YH205Y K213T Q360S D416V R437W H133Y 20 73 K176L F201Y H205Y K213T Q360SD416V R437W <1 40 H142W K176L E185P 22 74 K176L E185P L318W 18 65 *2aHY158W K176L E185P F201Y H205Y D207V V209D H316W 25 107 *2aH K176L E185P36 69 *2aH H68W K176L E185P H133Y 53 159 *2aH K176L E185P R437W 35 64*2aH K176L E185P H316W R437W 43 75 *2aH K176L E185P H316W R437W L318W 4076 *2aH K176L E185P H316W R437W H133Y 52 102 *2aH K176L E185P H316WR437W Y156W Y158W 35 72 *2aH K176L E185P H316W R437W H142W 43 91 *2aHK176L E185P R437W Q360S 41 78 *2aH K176L E185P R437W Q360S Y156W 38 69*2aH K176L E185P R437W Q360S H142W 31 71 *2aH K176L E185P R437W Q360SH133Y 44 78 *2aH K176L E185P R437W Q360S H316W 38 85 *2aH K176L E185PH316W R437W Q360S 41 75 K176L F201Y H205Y K213T Q360S D416V R437W E185P37 106 K176L F201Y H205Y K213T Q360S D416V R437W E185P H142W 39 91 K176LF201Y H205Y K213T Q360S D416V R437W E185P H68W 36 105 K176L F201Y H205YK213T Q360S D416V R437W E185P Y158W H133Y 42 67 K176L F201Y H205Y K213TQ360S D416V R437W E185P Y158W H142W 40 85 K176L F201Y H205Y K213T Q360SD416V R437W E185P Y158W Y156W 35 80 K176L F201Y H205Y K213T Q360S D416VR437W E185P Y158W H316W 50 108 K176L F201Y H205Y K213T Q360S D416V R437WE185P Y158W H316L L318W 27 42 *2aH H68W K176L E185P 32 86 *2aH H68WK176L E185P Y158W 28 77 *2aH H68W K176L E185P Y156W 28 64 *2aH H68WK176L E185P F279W 32 121 *2aH H68W K176L E185P F201Y H205Y D207V V209D22 33 *2aH H68W K176L E185P H133Y 43 >280

Example 3 Improved Liquefaction Performance of Alpha-Amylase Variants atpH 4.5

A pilot plant scale liquefaction was set up to achieve the starchliquefaction, and a Neocuproine assay was set up to measure the dextroseequivalent (DE) to evaluate the performance of liquefaction.

Starch slurry with final 5.5 ppm Calcium concentration and 34-35% Drysolids content (DS), was prepared in the tank.

Preparation of Starch Slurry:

Approximately 60 L of distilled water was added to the 200 L slurrytank. The agitator was turned on and dust collection system activatedfor removal of starch dust. Two 50-lb bags of starch (90.39% DS) wereadded to the mixing water. After all starch was added, a paddle was usedto get settled starch moving and keep the entire slurry mixing. Thedensity of the slurry mixture was measured with a hydrometer in a 2 Lgraduated cylinder. The density was decreased by stepwise addition ofdistilled water until approximately 1.155 g/mL, near 35% DS.

pH and conductivity probes were immersed in the mixing slurry. pH wasadjusted to pH 4.5 (variants) or pH 5.5 (Liquozyme X) with either 1M HClor 2M NaOH. The starch contains 16 ppm Calcium thus final calciumconcentration in slurry is 5.5 ppm with 34-35% DS. Conductivity wasaround 250 μS/cm.

Enzyme at the appropriate dose was added just before running the slurrythrough the jet.

Variants were dosed at 4.69 micrograms per gram DS and iUquozyme X(reference) was dosed at 75 NU(T)/g DS:

-   -   Enzymes with known activity (KNU(T)/g) were dosed as NU(T) per g        DS (dilution is weight per volume):

${{Enzyme}\left( {µ\; l} \right)} = {{\frac{{{{Starch}(g)} \cdot \%}\mspace{14mu}{{DS}_{Starch} \cdot {{Dose}\left( \frac{{NU}(T)}{g{DS}} \right)}}}{{Enzyme}\mspace{14mu}{{{activity}\left( \frac{{NU}(T)}{g{DS}} \right)} \cdot {{Dilution}\left( \frac{g}{mL} \right)}}} \cdot 1000}\frac{µ\; l}{mL}}$

-   -   Enzymes with known protein concentration (ug EP/mL) were dosed        as ug enzyme protein per g DS (dilution is volume per volume):

${{Enzyme}\left( {µ\; l} \right)} = {{\frac{{{{Starch}(g)} \cdot \%}\mspace{14mu}{{DS}_{Starch} \cdot {{Dose}\left( \frac{µ\; g}{g{DS}} \right)}}}{{Enzyme}\mspace{14mu}{{{Conc}\left( \frac{mg}{mL} \right)} \cdot {{Dilution}\left( \frac{mL}{mL} \right)}}} \cdot 1000}\frac{µ\; l}{mL}}$

Slurry was pumped to the jet and held for 5 minutes at around 105° C. asthe primary liquefaction. After 10 minutes (approximately 2 retentiontimes) a sample was taken out.

After that, liquefied starch was transferred to the oil bath at 95-97°C. for additional 15, 30, 45, 60, 75 and 90 minutes as the secondaryliquefaction.

After primary and secondary liquefaction, starch was hydrolyzedintomaltodextrins, during which reducing sugars were liberated.

Copper ions were reduced by the reducing sugars and reacted withNeocuproine reagent to form a colored complex, which gave the content ofthe reducing sugars in the liquefacts. This value was reported as thedextrose equivalent (DE) as indication of the degree of liquefaction.The higher the DE value is, the more advanced liquefaction has beenachieved.

A volumetric flask (500 mL) was prepared for each sample by addingapproximately 250 mL DDI water and 400 μl 1M HCl. With each liquefactsample, the flask was tared and approximately 0.50 g of sample was addedto the flask, recording the weight. Solids content of the 90 min samplewas determined from RI analysis. DE measurements were performed induplicates.

The flasks were brought to volume with DDI water and stir bars wereadded. After inverting several times to mix, the flasks were placed on amulti-point magnetic stir plate.

Method for Dextrose Equivalents (DE)

Dextrose equivalents (DE) were assayed as described below.

Place 200 μl of the above samples from the flask to 15 ml conical tube.Add 0.8 ml of reagent A and B each (made according to the compositionwritten below).

Reagent A:

Add 40 g Na₂CO₃, 16 g of Glycine, 0.45 g of CuSO₄.5H₂O to 800 ml MQwater in measuring flask and make up the final volume to 1000 ml.Aliquot into 50 ml tubes and wrap in aluminum foil and keep it in thedark.

Reagent B:

Add 1.2 g Neocuproine to 1000 ml MQ water. Aliquot into 50 ml tubes andwrap in aluminum foil and keep it in the dark.

Similar additions are made for the standards and blank (just water) aswell. Put them all in the flask stand. Boil the samples for 12 minutes.Stop the reaction immediately by putting all the samples in the icewater. After cooling the samples, add 3.2 ml Milli-Q water. After mixingthe samples, measure the OD at 450 nm Create a standard curve from ODreadings of glucose standards.

The slope and intercept of the standard curve was used to calculate thereducing end concentrations in the liquefaction samples. The R² of thestandard curve was used to assess the quality of the DE method. Theblank were used to determine if the Neocuproine reagents needed to bereplaced.

All absorbances were corrected by subtracting the blanks:Corrected OD₄₅₀=OD_(450,Actual)−OD₄₅₀,Blinds

The concentration of dextrin reducing ends in the sample was calculatedfrom the calibration curve. The RI value was converted to an equivalent% DS by interpolating a DE12 table. The sample DE was calculated:

${DE} = {\frac{{Reducing}\mspace{14mu}{{ends}\left( \frac{µ\; g}{mL} \right)}}{{{{Sample}(g)} \cdot \%}\mspace{14mu}{{DS} \cdot \frac{10^{6}\mspace{14mu} µ\; g}{1\mspace{14mu} g} \cdot \frac{1}{{Dilution}\;(X)}}} \times 100\%}$

Results in Table 6 showed that all variants gave higher average DEvalues at pH 4.5 than Liquozyme X at pH 5.5, indicating more efficientand advanced liquefaction.

TABLE 6 Improved dextrose equivalent (DE) of alpha-amylase variants atpH 4.5 compared to Liquozyme X (reference) at pH 5.5 at around 250μS/cm, 5.5 ppm Calcium and 34-35% DS. All the variants are variants ofLiquozyme X. All results are the average of at least two determinations.DE Mutations pH 15 min 30 min 45 min 60 min 75 min 90 min Reference 5.52.6 3.8 5.2 6.5 7.5 8.8 K176L 4.5 3.0 4.7 6.3 7.9 9.0 9.9 F201Y H205YK213T Q360S D416V R437W E185P K176L 4.5 3.7 5.7 7.8 9.9 10.9 12.4 F201YH205Y K213T Q360S D416V R437W E185P H68W K176L 4.5 3.2 5.1 6.6 8.5 9.610.4 F201Y H205Y K213T Q360S D416V R437W E185P H142W K176L 4.5 1.8 3.34.2 5.3 6.0 6.7 F201Y H205Y K213T Q360S D416V R437W E185P Y158W *2aH 4.52.5 4.1 5.7 7.1 8.4 9.3 K176L E185P R437W Q360S H133Y K176L 4.5 3.6 5.57.0 8.5 10.4 10.9 F201Y H205Y K213T Q360S D416V R437W E185P Y158W H133YK176L 4.5 2.9 4.6 6.2 7.7 9.2 9.8 F201Y H205Y K213T Q360S D416V R437WE185P Y158W H142W

Example 4 Improved Small Scale Liquefaction Performance of Alpha-AmylaseVariants at pH 4.5 and pH 4.8

Small scale liquefaction assay was set up to achieve the starchliquefaction, and a Neocuproine assay was set up to measure the dextroseequivalent (DE) to evaluate the performance of liquefaction.

Low calcium starch (90.32% DS) was weighed out and mixed with DDI waterto prepare a 40 g slurry at 33.25% DS. Calcium was added such that thetotal calcium content was 5 ppm (5 mg/L). Assuming a diluted enzyme andCaCl₂ density of 1.0 g/mL, this volume was calculated by:

${0.25M\mspace{14mu}{{CaCl}_{2\mspace{14mu}}\left( {µ\; l} \right)}} = \frac{{Total}\mspace{14mu}{{{slurry}(g)} \cdot {{Ca}^{++}\left( \text{mg/L} \right)}}\frac{{Enzyme}(g)}{Dilution}{{Ca}_{Enzyme}^{++}\left( \text{mg/L} \right)}}{40.08\frac{g\mspace{14mu}{Ca}^{++}}{{mol}\mspace{14mu}{CaCl}_{2}}0.2495\frac{{mol}\mspace{14mu}{CaCl}_{2}}{L}}$

Enzyme was added next. Assuming an enzyme density of 1.0 g/mL:

-   -   Enzymes with known activity (KNU(T)/g) are dosed as NU(T) per g        DS:

${{Enzyme}\left( {µ\; l} \right)} = {{\frac{{{{Starch}(g)} \cdot \%}\mspace{14mu}{{DS}_{Starch} \cdot {{Dose}\left( \frac{{NU}(T)}{g{DS}} \right)}}}{{Enzyme}\mspace{14mu}{{{A{ctivity}}\left( \frac{{NU}(T)}{g} \right)} \cdot {{Dilution}(X)}}} \cdot 1}{\frac{g}{mL} \cdot 1000}\frac{µ\; l}{mL}}$

-   -   Enzymes with known protein concentration (μg EP/mL) are dosed as        ug enzyme protein per g DS:

${{Enzyme}\left( {µ\; l} \right)} = {{\frac{{{{Starch}(g)} \cdot \%}\mspace{14mu}{{DS}_{Starch} \cdot {{Dose}\left( \frac{µ\; g}{g{DS}} \right)}}}{{Enzyme}\mspace{14mu}{{{Conc}\left( \frac{µ\; g}{mL} \right)} \cdot {{Dilution}(X)}}} \cdot 1000}\frac{µ\; l}{mL}}$

Often, the enzyme addition would raise the pH by 0.1 or so. The initialpH of the starch slurry was near 4.78. pH was adjusted to pH 4.8 with 1M HCl as necessary or to pH 4.5 with NaOH. Conductivity was adjustedwith 1 M NaCl to 200 μS/cm. Finally, DDI water was added so that thefinal solids content of the slurry was 32%. A repeater pipette was thenused to aliquot 3.3 mL of starch slurry to each vial. Vials were cappedand placed in the JKem shaker block.

The shaking block is heated to target temperature for primary step inabout 12 minutes. The vials are placed simultaneously in the heatingblock and the shaking starts. It takes 1 minute for the starch slurry toreach the target temperature of 105.5° C. After 6 minutes, a valve isturned on manually to allow a cooling liquid to pass through the heatingblock to drop the temperature to 95° C. and the temperature controlleris adjusted to 95° C. It takes about 1 minute for the temperature instarch slurry to stabilize at the secondary liquefaction step. Thereaction continues at 95° C. for 90 minutes. One vial is removed every15 minutes for DE measurement, as described above.

TABLE 7 Improved dextrose equivalent (DE) of variants compared toLiquozyme X (reference) in small scale liquefaction assay at pH 4.8, 200μS/cm, 5 ppm Calcium and enzyme dosage of 4.69 μg enzyme protein/g DS.All the variants are variants of Liquozyme X. All results are theaverage of at least two determinations. DE Mutations pH 15 min 30 min 45min 60 min 75 min 90 min Reference 4.8 1.6 2.1 2.4 2.8 3.0 3.7 S239A 4.81.7 2.5 3.1 3.1 3.5 3.2 K176L 4.8 2.7 4.0 4.8 5.7 6.8 7.2 *2aW *2bHK176L E185P 4.8 4.1 8.3 11.6 12.3 *2aH *2bW K176L E185P 4.8 4.2 7.4 11.112.7 *2aH K176L E185P 4.8 4.2 8.0 11.8 12.5 G71W K176L E185P 4.8 2.6 3.64.9 5.6 7.1 8.4 K176L E185P 4.8 4.3 6.3 8.2 9.5 10.9 12.1 E185P 4.8 2.63.9 5.0 6.4 6.9 8.3 K176L F201Y H205Y K213T 4.8 5.6 8.4 10.6 12.7 13.414.9 Q360S D416V R437W K176L 4.8 2.8 4.2 5.9 6.2 8.3 8.8

TABLE 8 Improved dextrose equivalent (DE) of variants compared toLiquozyme X (reference) in small scale liquefaction assay at pH 4.8, 200μS/cm, 5 ppm Calcium and enzyme dosage of 3.127 μg enzyme protein/g DS.All the variants are variants of Liquozyme X. All results are theaverage of at least two determinations. DE 30 45 60 75 90 Mutations pH15 min min min min min min K176L F201Y H205Y 4.8 3.1 4.7 5.6 7.2 8.4 8.6K213T Q360S D416V R437W K176L F201Y H205Y 4.8 13.8 K213T Q360S D416VR437W E185P H68W K176L F201Y H205Y 4.8 2.7 3.7 3.9 4.6 5.6 7.3 K213TQ360S D416V R437W L318W K176L F201Y H205Y 4.8 2.9 3.9 4.4 5.9 6.4 7.2K213T Q360S D416V R437W H316W K176L F201Y H205Y 4.8 3.3 4.8 5.8 7.7 8.89.6 K213T Q360S D416V R437W H133Y K176L F201Y H205Y 4.8 8.4 K213T Q360SD416V R437W H142W K176L F201Y H205Y 4.8 6.6 K213T Q360S D416V R437WY156W K176L F201Y H205Y 4.8 7.3 K213T Q360S D416V R437W Y158W K176LF201Y H205Y 4.8 6.8 K213T Q360S D416V R437W H68W *2aH K176L F201Y 4.83.0 4.4 5.6 7.1 7.3 8.9 H205Y K213T Q360S D416V R437W K176L F201Y H205Y4.8 1.3 2.5 1.8 2.2 2.6 2.7 K213T Q360S D416V R437W N126W

TABLE 9 Improved dextrose equivalent (DE) of variants compared toLiquozyme X (reference) in small scale liquefaction assay at pH 4.5, 200μS/cm, 5 ppm Calcium and enzyme dosage of 4.69 μg enzyme protein/g DS.All the variants are variants of Liquozyme X. All results are theaverage of at least two determinations. DE Mutations pH 15 min 30 min 45min 60 min 75 min 90 min Reference 5.5 3.3 5.1 6.5 8.0 9.2 10.3 K176LF201Y H205Y K213T Q360S 4.5 2.8 5.0 5.0 5.3 6.1 5.6 D416V R437W K176LE185P 4.5 3.2 5.4 5.9 7.4 8.4 9.0 H142W K176L E185P 4.5 3.3 6.0 8.7 8.3H68W K176L E185P 4.5 3.0 5.4 7.0 7.1 Y158W K176L E185P 4.5 3.0 5.8 8.68.8 K176L E185P F279W 4.5 2.9 5.0 6.2 7.2 *2aH K176L E185P 4.5 3.5 6.28.3 8.8 K176L E185P H316W 4.5 3.3 6.2 9.5 9.9 *2aH *2bW K176L E185P 4.53.3 5.1 7.7 7.5 K176L E185P L318W 4.5 3.3 5.9 6.5 8.5 *2aW *2bH K176LE185P 4.5 3.9 6.1 9.0 8.8 K176L F201Y H205Y Q360S D416V 4.5 2.7 5.6 5.56.8 R437W K176L F201Y H205Y K213T D416V 4.5 2.3 3.1 3.8 3.9 R437W K176LF201Y H205Y K213T Q360S 4.5 2.0 4.1 3.9 7.0 R437W K176L F201Y H205YK213T Q360S 4.5 5.6 9.2 11.9 12.4 14.3 15.1 D416V R437W E185P *2aH H68WK176L E185P Y156W 4.5 2.7 5.6 7.0 7.5 *2aH H68W K176L E185P F279W 4.52.9 4.7 6.3 7.1 7.7 9.1 *2aH H68W K176L E185P H133Y 4.5 3.3 6.4 9.1 10.3K176L F201Y H205Y K213T Q360S 4.5 5.9 9.1 11.4 12.8 14.3 15.0 D416VR437W E185P H68W K176L F201Y H205Y K213T Q360S 4.5 6.0 9.8 10.6 13.114.6 14.5 D416V R437W E185P H142W K176L F201Y H205Y K213T Q360S 4.5 5.210.1 12.9 13.4 14.4 D416V R437W E185P Y158W *2aA *2bH K176L E185P H316WL318W 4.5 3.3 6.9 7.6 9.5 11.0 R437W *2aA *2bH H133Y K176L E185P H316W4.5 3.9 7.4 9.1 9.9 10.7 R437W Y156W Y158W K176L E185P H316W 4.5 1.8 3.14.3 5.3 R437W H133Y Y158W K176L E185P F201Y 4.5 5.8 11.1 13.8 15.8 H205YK213T Q360S D416V R437W Y158W K176L E185P F201Y H205Y 4.5 3.3 6.3 7.29.1 9.6 D207V V209D H316W Y156W K176L E185P Q360S R437W 4.5 4.4 8.6 9.711.8 12.6 *2aH K176L E185P R437W Q360S H133Y 4.5 7.8 13.3 16.4 18.0 20.2*2aH K176L E185P R437W Q360S H316W 4.5 5.4 10.2 10.6 12.8 13.1 K176LE185P F201Y H205Y R437W 4.5 1.7 3.3 4.6 4.9

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The invention claimed is:
 1. An alpha-amylase variant, comprising a substitution at position 176 with L and a substitution at position 201 with Y, wherein (a) each position corresponds to the positions of SEQ ID NO: 1; (b) the variant has at least 90% and less than 100% sequence identity to SEQ ID NO: 14; and (c) the variant has alpha-amylase activity.
 2. The alpha-amylase variant of claim 1, which has at least 95% and less than 100% sequence identity to SEQ ID NO:
 14. 3. The alpha-amylase variant of claim 1, further comprising a substitution at one or more positions corresponding to positions 68, 71, 126, 133, 142, 144, 156, 158, 185, 205, 213, 239, 279, 316, 318, 360, 416, 437 and 450 of SEQ ID NO:
 1. 4. The alpha-amylase variant of claim 1, further comprising a substitution at a position corresponding to position 68 of SEQ ID NO:
 1. 5. The alpha-amylase variant of claim 4, wherein the substitution at a position corresponding to position 68 is 68F, 68Y, or 68W.
 6. The alpha-amylase variant of claim 1, further comprising a substitution at a position corresponding to position 185 of SEQ ID NO:
 1. 7. The alpha-amylase variant of claim 6, wherein the substitution at a position corresponding to position 185 is 185P.
 8. The alpha-amylase variant of claim 1, further comprising a substitution at a position corresponding to position 205 of SEQ ID NO:
 1. 9. The alpha-amylase variant of claim 8, wherein the substitution at a position corresponding to position 205 is 205Y.
 10. The alpha-amylase variant of claim 1, further comprising a substitution at a position corresponding to position 213 of SEQ ID NO:
 1. 11. The alpha-amylase variant of claim 10, wherein the substitution at a position corresponding to position 213 is 213T.
 12. The alpha-amylase variant of claim 1, further comprising a substitution at a position corresponding to position 360 of SEQ ID NO:
 1. 13. The alpha-amylase variant of claim 12, wherein the substitution at position 360 is 360S.
 14. The alpha-amylase variant of claim 1, further comprising a substitution at a position corresponding to position 416 of SEQ ID NO:
 1. 15. The alpha-amylase variant of claim 14, wherein the substitution at a position corresponding to position 416 is 416V.
 16. The alpha-amylase variant of claim 1, further comprising a substitution at a position corresponding to position 437 of SEQ ID NO:
 1. 17. The alpha-amylase variant of claim 16, wherein the substitution at a position corresponding to position 437 is 437W.
 18. The alpha-amylase variant of claim 1, selected from the group consisting of: 68W+176L+185P+201Y+205Y+213T+360S+416V+437W, 68W+176L+201Y+205Y+213T+360S+416V+437W, 126W+176L+201Y+205Y+213T+360S+416V+437W, 133Y+158W+176L+185P+201Y+205Y+213T+360S+416V+437W, 133Y+176L+201Y+205Y+213T+360S+416V+437W, 142W+158W+176L+185P+201Y+205Y+213T+360S+416V+437W, 142W+176L+185P+201Y+205Y+213T+360S+416V+437W, 142W+176L+201Y+205Y+213T+360S+416V+437W, 156W+158W+176L+185P+201Y+205Y+213T+360S+416V+437W, 156W+176L+201Y+205Y+213T+360S+416V+437W, 158W+176L+185P+201Y+205Y+207V+209D+316W, 158W+176L+185P+201Y+205Y+213T+316L+318W+360S+416V+437W, 158W+176L+185P+201Y+205Y+213T+316W+360S+416V+437W, 158W+176L+185P+201Y+205Y+213T+360S+416V+437W, 158W+176L+201Y+205Y+213T+360S+416V+437W, 176L+185P+201Y+205Y+213T+360S+416V+437W, 176L+185P+201Y+205Y+437W, 176L+201Y+205Y+213T+239Q+360S+416V+437W, 176L+201Y+205Y+213T+316W+360S+416V+437W, 176L+201Y+205Y+213T+318W+360S+416V+437W, 176L+201Y+205Y+213T+360S+416V+437W, 176L+201Y+205Y+213T+360S+437W, 176L+201Y+205Y+213T+416V+437W, and 176L+201Y+205Y+360S+416V+437W.
 19. A detergent composition comprising the alpha-amylase variant of claim 1 and a surfactant.
 20. A method of cleaning a hard surface or a textile, comprising contacting the hard surface or the textile with the alpha-amylase variant of claim
 1. 21. A method of producing liquefied starch, comprising liquefying a starch-containing material with the alpha-amylase variant of claim
 1. 22. A method of producing a fermentation product, comprising (a) liquefying a starch-containing material with the alpha-amylase variant of claim 1 to produce a liquefied mash; (b) saccharifying the liquefied mash to produce fermentable sugars; and (c) fermenting the fermentable sugars in the presence of a fermenting organism.
 23. An isolated polynucleotide encoding the alpha-amylase variant of claim
 1. 24. A nucleic acid construct or expression vector comprising the polynucleotide of claim 23, wherein the polynucleotide is operably linked to one or more control sequences that direct the production of the polypeptide in a host cell.
 25. A host cell comprising the nucleic acid construct or expression vector of claim
 24. 26. A method of producing an alpha-amylase variant, comprising: (a) cultivating the host cell of claim 25 under conditions suitable for expression of the variant; and (b) recovering the alpha-amylase variant. 